Alpha-fetoprotein Immu31 antibodies and fusion proteins and methods of use thereof

ABSTRACT

The present invention provides humanized, chimeric and human anti-alpha-fetoprotein antibodies, fusion proteins, and fragments thereof. The antibodies, fusion proteins, and fragments thereof, as well as combinations with other suitable antibodies, are useful for the treatment and diagnosis of hepatocellular carcinoma, hepatoblastoma, germ cell tumors carcinoma and other AFP-producing tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/399,707, filed Aug. 1 2002, which is incorported herein byreference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

[0002] The present invention relates to humanized, chimeric and humanalpha-fetoprotein (AFP) antibodies, particularly therapeutic anddiagnostic conjugates of humanized, chimeric and human forms. Inparticular, the invention includes Immu31 antibodies and methods oftreating hepatocellular carcinoma, germ cell tumors, and other AFP.producing tumors using humanized, chimeric and human antibody forms. Thepresent invention also relates to antibody fusion proteins or fragmentstherof comprising at least two Immu31 MAbs or fragments thereof or atleast one Immu31 MAb or fragment therof and at least one second MAb orfragment therof, other than the Immu31 MAb or fragment thereof. Thehumanized, chimeric and human Immu31 MAbs, fragments therof and antibodyfusion proteins thereof, or fragments thereof, may be administeredalone, conjugated to diagnostic and/or therapeutic agents, incombination with a therapeutic or diagnostic immunoconjugate, incombination with other naked antibodies, or with at least onetherapeutic agent and/or diagnostic agent. The present invention furthercontemplates DNA sequences encoding humanized, chimeric and human Immu31antibodies and fragments thereof, antibody fusion proteins and fragmentsthereof, vectors and host cells containing the DNA sequences, andmethods of making the humanized, chimeric and human Immu31 antibodies.that produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

[0003] MAbs can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

[0004] Abs to peptide backbones are generated by well-known methods forAb production. For example, injection of an immunogen, such as(peptide)_(n)-KLH, wherein KLH is keyhole limpet hemocyanin, and n=1-30,in complete Freund's adjuvant, followed by two subsequent injections ofthe same immunogen suspended in incomplete Freund's adjuvant intoimmunocompetent animals. The animals are given a final i.v. boost ofantigen, followed by spleen cell harvesting three days later. Harvestedspleen cells are then fused with Sp2/0-Ag14 myeloma cells and culturesupernatants of the resulting clones analyzed for anti-peptidereactivity using a direct-binding ELISA. Fine specificity of generatedAbs can be analyzed for by using peptide fragments of the originalimmunogen. These fragments can be prepared readily using an automatedpeptide synthesizer. For Ab production, enzyme-deficient hybridomas areisolated to enable selection of fused cell lines. This technique alsocan be used to raise antibodies to one or more of the chelatescomprising the linker, e.g., In(III)-DTPA chelates. Monoclonal mouseantibodies to an In(III)-di-DTPA are known (Barbet '395 supra).

[0005] After the initial raising of antibodies to the immunogen, thevariable genes of the monoclonal antibodies can be cloned from thehybridoma cells, sequenced and subsequently prepared by recombinanttechniques. Humanization and chimerization of murine antibodies andantibody fragments are well known to those skilled in the art. Forexample, humanized monoclonal antibodies are produced by transferringmouse complementary determining regions from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen, substituting human residues in the framework regions of the murinecounterparts. In a preferred embodiment, some human residues in theframework regions of the humanized anti-AFP antibody or fragmentsthereof are replaced by their murine counterparts. Preferably, thehumanized anti-AFP antibody is a humanized Immu31 antibody. It is alsopreferred that a combination of framework sequences from 2 differenthuman antibodies are used for V_(H). Still preferred, the two humanantibodies are EU and NEWM. The constant domains of the antibodymolecule is derived from those of a human antibody. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions.

[0006] General techniques for cloning murine immunoglobulin variabledomains are described, for example, by the publication of Orlandi etal., Proc. Nat'l Acad. Sci. U.S. Pat. No. 86: 3833 (1989), which isincorporated by reference in its entirety. Techniques for constructingchimeric antibodies are well known to those of skill in the art. As anexample, Leung et al., Hybridoma 13:469 (1994), describe how theyproduced an LL2 chimera by combining DNA sequences encoding the V_(K)and V_(H) domains of LL2 monoclonal antibody, an anti-CD22 antibody,with respective human κ and IgG₁ constant region domains. Thispublication also provides the nucleotide sequences of the LL2 light andheavy chain variable regions, V_(κ) and V_(H), respectively. Techniquesfor producing humanized MAbs are described, for example, by Jones etal., Nature 321: 522 (1986), Riechmann et al., Nature 332: 323 (1988),Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'lAcad. Sci. U.S.A 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437(1992), and Singer et al., J. Immun. 150: 2844 (1993), each of which ishereby incorporated by reference.

[0007] Another method for producing the antibodies of the presentinvention is by production in the milk of transgenic livestock. See,e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No.5,827,690, both of which are incoporated in their entirety by reference.Two DNA constructs are prepared which contain, respectively, DNAsegments encoding paired immunoglobulin heavy and light chains. The DNAsegments are cloned into expression vectors which contain a promotersequence that is preferentially expressed in mammary epithelial cells.Examples include, but are not limited to, promoters from rabbit, cow andsheep casein genes, the cow α-lactoglobulin gene, the sheepβ-lactoglobulin gene and the mouse whey acid protein gene. Preferably,the inserted fragment is flanked on its 3′ side by cognate genomicsequences from a mammary-specific gene. This provides a polyadenylationsite and transcript-stabilizing sequences. The expression cassettes arecoinjected into the pronuclei of fertilized, mammalian eggs, which arethen implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of bothtransgenes by Southern analysis. In order for the antibody to bepresent, both heavy and light chain genes must be expressed concurrentlyin the same cell. Milk from transgenic females is analyzed for thepresence and functionality of the antibody or antibody fragment usingstandard immunological methods known in the art. The antibody can bepurified from the milk using standard methods known in the art.

[0008] A chimeric antibody is a recombinant protein that contains thevariable domains including the CDRs derived from one species of animal,such as a rodent antibody, while the remainder of the antibody molecule;i.e., the constant domains, is derived from a human antibody.Accordingly, a chimeric monoclonal antibody (MAb) can also be humanizedby replacing the sequences of the murine FR in the variable domains ofthe chimeric MAb with one or more different human FR. Specifically,mouse CDRs are transferred from heavy and light variable chains of themouse immunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more human residues in the FR regions with their murinecounterparts to obtain an antibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239: 1534 (1988). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, WO0029584A1.

[0009] A fully human antibody of the present invention, i.e., a humananti-AFP MAb or another human antibody, such as anti-CEA, anti-TAG-72,anti-Tn, anti-Le(y), anti-MUC1, anti-MUC2, anti-MUC3, anti-MUC4,anti-EGFR, anti-HER2 and anti-TNF (tumor necrosis factor) used forcombination therapy with humanized or chimeric Immu31 antibodies, can beobtained from a transgenic non-human animal. See, e.g., Mendez et al.,Nature Genetics, 15: 146-156 (1997) and U.S. Pat. No. 5,633,425, whichare incoporated in their entirety by reference. For example, a humanantibody can be recovered from a transgenic mouse possessing humanimmunoglobulin loci. Preferably, the anti-AFP antibody isan Immu31antibody. The mouse humoral immune system is humanized by inactivatingthe endogenous immunoglobulin genes and introducing human immunoglobulinloci. The human immunoglobulin loci are exceedingly complex and comprisea large number of discrete segments which together occupy almost 0.2% ofthe human genome. To ensure that transgenic mice are capable ofproducing adequate repertoires of antibodies, large portions of humanheavy- and light-chain loci must be introduced into the mouse genome.This is accomplished in a stepwise process beginning with the formationof yeast artificial chromosomes (YACs) containing either human heavy- orlight-chain immunoglobulin loci in germline configuration. Since eachinsert is approximately 1 Mb in size, YAC construction requireshomologous recombination of overlapping fragments of the immunoglobulinloci. The two YACs, one containing the heavy-chain loci and onecontaining the light-chain loci, are introduced separately into mice viafusion of YAC-containing yeast spheroblasts with mouse embryonic stemcells. Embryonic stem cell clones are then microinjected into mouseblastocysts. Resulting chimeric males are screened for their ability totransmit the YAC through their germline and are bred with mice deficientin murine antibody production. Breeding the two transgenic strains, onecontaining the human heavy-chain loci and the other containing the humanlight-chain loci, creates progeny which produce human antibodies inresponse to immunization.

[0010] Unrearranged human immunoglobulin genes also can be introducedinto mouse embryonic stem cells via microcell-mediated chromosometransfer (MMCT). See, e.g., Tomizuka et al., Nature Genetics, 16: 133(1997). In this methodology microcells containing human chromosomes arefused with mouse embryonic stem cells. Transferred chromosomes arestably retained, and adult chimeras exhibit proper tissue-specificexpression.

[0011] As an alternative, an antibody or antibody fragment of thepresent invention may be derived from human antibody fragments isolatedfrom a combinatorial immunoglobulin library. See, e.g., Barbas et al.,METHODS. A Companion to Methods in Enzymology 2: 119 (1991), and Winteret al., Ann. Rev. Immunol. 12: 433 (1994), which are incorporated byreference. Many of the difficulties associated with generatingmonoclonal antibodies by B-cell immortalization can be overcome byengineering and expressing antibody fragments in E. coli, using phagedisplay. To ensure the recovery of high affinity, monoclonal antibodiesa combinatorial immunoglobulin library must contain a large repertoiresize. A typical strategy utilizes MRNA obtained from lymphocytes orspleen cells of immunized mice to synthesize cDNA using reversetranscriptase. The heavy- and light-chain genes are amplified separatelyby PCR and ligated into phage cloning vectors. Two different librariesare produced, one containing the heavy-chain genes and one containingthe light-chain genes. Phage DNA is islolated from each library, and theheavy-and light-chain sequences are ligated together and packaged toform a combinatorial library. Each phage contains a random pair ofheavy- and light-chain cDNAs and upon infection of E. coli directs theexpression of the antibody chains in infected cells. To identify anantibody that recognizes the antigen of interest, the phage library isplated, and the antibody molecules present in the plaques aretransferred to filters. The filters are incubated with radioactivelylabeled antigen and then washed to remove excess unbound ligand. Aradioactive spot on the autoradiogram identifies a plaque that containsan antibody that binds the antigen. Cloning and expression vectors thatare useful for producing a human immunoglobulin phage library can beobtained, for example, from STRATAGENE Cloning Systems (La Jolla,Calif.).

[0012] Further, recent methods for producing bispecific MAbs includeengineered recombinant MAbs which have additional cysteine residues sothat they crosslink more strongly than the more common immunoglobulinisotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225,1997. Another approach is to engineer recombinant fusion proteinslinking two or more different single-chain antibody or antibody fragmentsegments with the needed dual specificities. See, e.g., Coloma et al.,Nature Biotech. 15:159-163, 1997. A variety of bispecific fusionproteins can be produced using molecular engineering. In one form, thebispecific fusion protein is monovalent, consisting of, for example, ascFv with a single binding site for one antigen and a Fab fragment witha single binding site for a second antigen. In another form, thebispecific fusion protein is divalent, consisting of, for example, anIgG with two binding sites for one antigen and two scFv with two bindingsites for a second antigen.

[0013] Bispecific fusion proteins linking two or more differentsingle-chain antibodies or antibody fragments are produced in similarmanner. Recombinant methods can be used to produce a variety of fusionproteins. For example a fusion protein comprising a Fab fragment derivedfrom a humanized monoclonal Immu31 antibody and a scFv derived from amurine anti-diDTPA can be produced. A flexible linker, such as GGGSconnects the scFv to the constant region of the heavy chain of theImmu31 antibody. Alternatively, the scFv can be connected to theconstant region of the light chain of another humanized antibody.Appropriate linker sequences necessary for the in-frame connection ofthe heavy chain Fd to the scFv are introduced into the VL and VK domainsthrough PCR reactions. The DNA fragment encoding the scFv is thenligated into a staging vector containing a DNA sequence encoding the CH1domain. The resulting scFv-CH1 construct is excised and ligated into avector containing a DNA sequence encoding the V_(H) region of anImrnmu31 antibody. The resulting vector can be used to transfect anappropriate host cell, such as a mammalian cell for the expression ofthe bispecific fusion protein.

[0014] Preparation of Chimeric, Humanized and Human Anti-AFP Antibodies

[0015] Cell lines and culture media used in the present inventioninclude Immu31 hybridoma cells and Sp2/0-Ag14 myeloma cells (ATCC,Rockville, Md.). The monoclonal hybridoma producing Immu31 was obtainedby fusing the spleen cells prepared from a mouse that had been immunizedwith alpha-fetoprotein with SP2/0Ag14. These cells may be cultured inHybridoma serum-free media (HSFM) (life Technologies, Grand Island,N.Y.) supplemented with 10% fetal bovine serum (FBS) (HycloneLaboratories, Logan, Utah.) and antibiotics (complete media).Alternatively, they may be cultured in Dulbecco's modified Eagle'sMedium (DMEM) supplemented with 10% FCS (Gibco/BRL, Gaithersburg, Mass.)containing 10% of FCS and 75 μg/ml gantamicin (complete HSFM) or, whereindicated, in HSFM containing only antibiotics. Selection of thetransfectomas may be carried out in complete HSFM containing 500units/ml of hygromycin (Calbiochem, San Diego, Calif.). All cell linesare preferably maintained at 37° C. in 5% CO₂.

[0016] Obtaining Vκ and V_(H) Gene Segments

[0017] Isolation of the Vκ and V_(H) gene segments can be accomplishedby several means that are well-known in the art. Two such means include,but are not limited to, PCR cloning and cDNA library screening.

[0018] PCR cloning techniques are well-known in the art. In brief,however, PCR cloning of Vκ and V_(H) gene fragments may be accomplishedas follows. Total RNA may be isolated from a Immu31 hybridoma cell lineusing commercially available kits such as the Fast Track RNA Isolationkit (Invitrogen, San Diego, Calif.). The first strand cDNA may then bereverse transcribed from RNA using a cDNA cycle kit (Invitrogen). Inthis process, 5 μg of total RNA is annealed to an oligo dT or randomhexamer primer, or a murine IgG CH1-specific primer or a murineCk-specific primer. Examples of such primers include CH1B (5′-ACA GTCACT GAG CTG G-3′) and Ck3-BH1 (5′-GCC GGA TCC TGA CTG GAT GGT GGG AAGATG GAT ACA-3′), respectively. The first strand cDNA may be used astemplates to amplify the V_(H) and Vκ sequences by PCR, as described byOrlandi et al. For the Vκ region, a primer pair such as VK1BACK (5′-GACATT CAG CTG ACC CAG TCT CCA-3′) and IgKC3′ (5′-CTC ACT GGA TGG TGG GAAGAT GGA TAC AGT TGG-3′) may be used. For the V_(H) region, a primer pairsuch as VH1BACK (5′-AGG T(C/G)(A/C) A(A/G)C TGC AG(C/G) AGT C(A/T)GG-3′) and CH1B may be used. After amplification, the Vκ and V_(H)fragments may then be gel-purified and cloned into a cloning vector suchas the TA cloning vector (Invitrogen) for sequence analyses by thedideoxytermination method. Sequences confirmed to be of immunoglobulinorigin may then be used to construct chimeric Ab expression vectorsusing methods described by Leung et al. (Hybridoma, 13:469 (1994)).

[0019] As a preferred alternative to isolating the Vκ and V_(H) genesegments by PCR cloning, cDNA library screening may be utilized. cDNAscreening methods also are well known in the art. In brief, however, acDNA library may be constructed from the mRNA extracted from the murineImmu31 hybridoma cells in pSPORT vector (Life Technologies). The firststrand cDNA may be synthesized by priming ply A RNA from Immu31hybridoma with an oligo dT primer-NotI adaptor (Life Technologies).After the second strand synthesis and attachment of SalI adaptors, thecDNA pool may be size fractionated through a cDNA size fractionationcolumn. Fractionated cDNA may then be ligated to pSPORT vector andsubsequently transformed into Escherichia coli DH5α. A library may thenbe plated, transferred to filters, and amplified.

[0020] Screening of the cDNA library may be accomplished byhybridization with labeled probes specific for the heavy and lightchains. For example [32-P]-labeled probes such as MUCH-1 (5′-AGA CTG CAGGAG AGC TGG GAA GGT GTG CAC-3′) for heavy chain and MUCK-1 (5′-GAA GCACAC GAC TGA GGC ACC TCC AGA TGT-3′) for light chain. Clones that arepositive on a first screening may be transferred to duplicate plates andscreened a second time with the same probes.

[0021] RNA isolation, cDNA synthesis, and amplification can be carriedout as follows. Total cell RNA can be prepared from a Immu31 hybridomacell line, using a total of about 10⁷ cells, according to Sambrook etal., (Molecular Cloning: A Laboratory Manual, Second ed., Cold SpringHarbor Press, 1989), which is incorporated by reference. First strandcDNA can be reverse transcribed from total RNA conventionally, such asby using the SuperScript preamplification system (Gibco/BRL,Gaithersburg, Md.). Briefly, in a reaction volume of 20 μl, 50 ng ofrandom hexamer primers can be annealed to 5 μg of RNAs in the presenceof 2 μl of 10X synthesis buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl,25 mM MgCl₂,1 mg/ml BSA], 1 μl of 10 mM dNTP mix, 2 μl of 0.1 M DTT, and200 units of SuperScript reverse transcriptase. The elongation step isinitially allowed to proceed at room temperature for 10 min followed byincubation at 42° C. for 50 min. The reaction can be terminated byheating the reaction mixture at 90° C. for 5 min.

[0022] Synthesizing and labeling the screening probes can beaccomplished by well-known means. Depending on the detection systemsutilized, probe labeling will vary. Many kits for this purpose arecommercially available. One method for 32-P labeling of oligonucleotidesincludes the use of with [γ-³²P]ATP (Amersham Arlington Heights, Ill.)and T4 polynucleotide kinase (New England Biolabs, Beverly, Mass.),followed by column purification.

[0023] Preparation of a Chimeric Anti-AFP Antibody

[0024] In general, to prepare chimeric anti-AFP MAb, V_(H) and V κchainsof a AFP antibody may be obtained by methods such as those describedabove and amplified by PCR. In a preferred embodiment, the chimericanti-AFP antibody is a Immu31 antibody. The Vκ PCR products may besubcloned into a pBR327 based staging vector (VKpBR) as described byleung et al., Hybridoma, 13:469 (1994). The V_(H) PCR products may besubcloned into a similar pBluescript-based staging vector (VHpBS). Thefragments containing the Vκ and V_(H) sequences, along with the promoterand signal peptide sequences, can be excised from the staging vectorsusing HindIII and BamHI restriction endonucleases. The Vκ fragments(about 600 bp) can be subcloned into a mammalian expression vector (forexample, pKh) conventionally. pKh is a pSVhyg-based expression vectorcontaining the genomic sequence of the human kappa constant region, anIg enhancer, a kappa enhancer and the hygromycin-resistant gene.Similarly, the about 800 bp V_(H) fragments can be subcloned into pG1g,a pSVgpt-based expression vector carrying the genomic sequence of thehuman IgG1 constant region, an Ig enhancer and the xanthine-guaninephosphoribosyl transferase (gpt) gene. The two plasmids may beco-transfected into mammalian cells, such as Sp2/0-Ag14 cells, byelectroporation and selected for hygromycin resistance. Coloniessurviving selection are expanded, and supernatant fluids monitored forproduction of cImmu31 MAb by an ELISA method. A transfection efficiencyof about 1-10×10⁶ cells is desirable. An antibody expression level ofbetween 0.10 and 2.5 μg/ml can be expected with this system.

[0025] Alternately, the Vκ and V_(H) expression cassettes can beassembled in the modified staging vectors, VKpBR2 and VHpBS2, excised asXbaI/BamHI and XhoI/BamHI fragments, respectively, and subcloned into asingle expression vector, such as pdHL2, as described by Gilles et al.J. Immunol. Methods 125:191 (1989), Losman et al., Clin. Cancer Res.5:3101 (1999) and in Losman et al., Cancer, 80:2660 (1997) for theexpression in Sp2/0-Ag14 cells. Another vector that is useful in thepresent invention is the GS-vector, as described in Barnes et al.,Cytotechnology 32:109-123 (2000), which is preferably expressed in theNS0 cell line and CHO cells. Other appropriate mammalian expressionsystems are described in Werner et al., Arzneim.-Forsch./Drug Res.48(II), Nr. 8, 870-880 (1998).

[0026] Preparation of a Humanized Anti-AFP Antibody

[0027] In a preferred embodiment, the humanized anti-AFP antibody is ahumanized Immu31 antibody. Once the sequences for the hImmu31Vκ andV_(H) domains are designed, CDR engrafting can be accomplished by genesynthesis using long synthetic DNA oligonucleotides as templates andshort oligonucleotides as primers in a PCR reaction. In most cases, theDNA encoding the Vκ or VH domain will be approximately 350 bp long. Bytaking advantage of codon degeneracy, a unique restriction site mayeasily be introduced, without changing the encoded amino acids, atregions close to the middle of the V gene DNA sequence. For example, atDNA nucleotide positions 169-174 (amino acid positions 56-57) for thehImmu31VH domain, a unique KpnI site can be introduced while maintainingthe originally designed amino acid sequence (see the sequence in FIG.5A). Two long non-overlapping single-stranded DNA oligonucleotides (˜150bp) upstream and downstream of the KpnI site can be generated byautomated DNA oligonucleotide synthesizer (Cyclone Plus DNA Synthesizer,Milligen-Biosearch). As the yields of full length DNA oligonucleotidesmay be expected to be low, they can be amplified by two pairs offlanking oligonucleotides in a PCR reaction. The primers can be designedwith the necessary restriction sites to facilitate subsequent sequenceassembly and subcloning. Primers for the oligonucleotides should containoverlapping sequence at the KpnI site so that the resultant PCR productscan be joined in-frame at the KpnI site to form a full length DNAsequence encoding the hImmu31 VH domain. The ligation of the PCRproducts for the oligos at the KpnI site and their subcloning into thePstII/BstEII sites of the staging vector, VHpBS, can be completed in asingle three-fragment ligation step. The subcloning of the correctsequence into VHpBS can be first analyzed by restriction digestionanalysis and subsequently conformed by sequencing reaction according toSanger et al., Proc. Natl. Acad. Sci. U.S. Pat. No. 74 5463 (1977).

[0028] The HindIII/BamHI fragment containing the Ig promoter, leadersequence and the hImmu31V_(H) sequence can be excised from the stagingvector and subcloned to the corresponding sites in a pSVgpt-basedvector, pG1g, which contains the genomic sequence of the human IgGconstant region, an Ig enhancer and a gpt selection marker, forming thefinal expression vector, hImmu31pG1g. Similar strategies can be employedfor the construction of the hImmu31Vκ sequence. The restriction sitechosen for the ligation of the PCR products for the longoligonucleotides can be NsiI in this case.

[0029] The DNA sequence containing the Ig promoter, leader sequence andthe hImmu31 Vκ sequence can be excised from the staging vector VKpBR bytreatment with BamHI/HindIII, and can be subcloned into thecorresponding sites of a pSVhyg-based vector, pKh, which contains thegenomic sequence of human kappa chain constant regions, a hygromycinselection marker, an Ig and a kappa enhancer, forming the finalexpression vector, hImmu31 pKh.

[0030] The two plasmids can be co-transfected into an appropriate cell,e.g., myeloma Sp2/0-Ag14, colonies selected for hygromycin resistance,and supernatant fluids monitored for production of hImmu31 antibodiesby, for example, an ELISA assay, as described below. Alternately, the Vκand VH expression cassettes can be assembled in the modified stagingvectors, VKpBR2 and VHpBS2, excised as XbaI/BamHI and XhoI/BamHIfragments, respectively, and subcloned into a single expression vector,such as pdHL2, as described by Gilles et al., J. Immunol. Methods125:191 (1989), Losman et al., Clin. Cancer Res. 5:3101 (1999) and inLosman et al., Cancer, 80:2660 (1997) for the expression in Sp2/0-Ag14cells. Another vector that is useful in the present invention is the GSvector, as described in Barnes et al., Cytotechnology 32:109-123 (2000),which is preferably expressed in the NSO cell line and CHO cells. Otherappropriate mammalian expression systems are described in Werner et al.,Arzneim.-Forsch./Drug Res. 48(11), Nr. 8, 870-880 (1998).

[0031] Transfection, and assay for antibody secreting clones by ELISA,can be carried out as follows. About 10 μg of hImmu31pKh (light chainexpression vector) and 20 μg of hImmu31pG1g (heavy chain expressionvector) can be used for the transfection of 5×10⁶ SP2/0 myeloma cells byelectroporation (BioRad, Richmond, Calif.) according to Co et al., J.Immunol., 148: 1149 (1992) which is incorporated by reference. Followingtransfection, cells may be grown in 96-well microtiter plates incomplete HSFM medium (GIBCO, Gaithersburg, Md.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 μg/ml of hygromycin. Colonies typically emerge 2-3weeks post-electroporation. The cultures can then be expanded forfurther analysis.

[0032] Screening the Clones and Isolating Antibodies

[0033] Transfectoma clones that are positive for the secretion ofchimeric or humanized heavy chain can be identified by ELISA assay.Briefly, supernatant samples (100 μl) from transfectoma cultures areadded in triplicate to ELISA microtiter plates precoated with goatanti-human (GAH)-IgG, F(ab′)₂ fragment-specific antibody (JacksonImmunoResearch, West Grove, Pa.). Plates are incubated for 1 h at roomtemperature. Unbound proteins are removed by washing three times withwash buffer (PBS containing 0.05% polysorbate 20). Horseradishperoxidase (HRP) conjugated GAH-IgG, Fc fragment-specific antibodies(Jackson ImmunoResearch, West Grove, Pa.) are added to the wells, (100μl of antibody stock diluted ×10⁴, supplemented with the unconjugatedantibody to a final concentration of 1.0 μg/ml). Following an incubationof 1 h, the plates are washed, typically three times. A reactionsolution, [100 μl, containing 167 μg of orthophenylene-diamine (OPD)(Sigma, St. Louis, Mo.), 0.025% hydrogen peroxide in PBS], is added tothe wells. Color is allowed to develop in the dark for 30 minutes. Thereaction is stopped by the addition of 50 μl of 4 N HCl solution intoeach well before measuring absorbance at 490 nm in an automated ELISAreader (Bio-Tek instruments, Winooski, Vt.). Bound chimeric antibodiesare than determined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

[0034] Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbancies at 280/260 nm. Peak fractions arepooled, dialyzed against PBS, and the antibody concentrated, forexample, with the Centricon 30 (Amicon, Beverly, Mass.). The antibodyconcentration is determined by ELISA, as before, and its concentrationadjusted to about 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), isconveniently added to the sample as preservative.

[0035] The affinity of a chimeric, humanized or human anti-AFP antibodymay be evaluated using a direct binding assay or a competitive bindingassay.

[0036] Modifying/Optimizing the Recombinant Antibodies

[0037] As humanization sometimes results in a reduction or even loss ofantibody affinity, additional modification might be required in order torestore the original affinity (See, for example, Tempest et al.,Bio/Technology 9: 266 (1991); Verhoeyen et al., Science 239: 1534(1988)), which are incorporated by reference. Knowing that cImmu31exhibits a binding affinity comparable to that of its murinecounterpart, defective designs, if any, in the original version ofhImm31u can be identified by mixing and matching the light and heavychains of cImmu31 to those of the humanized version. Preferably, somehuman residues in the framework regions are replaced by their murinecounterparts. Also preferred, a combination of framework sequences from2 different human antibodies, such as EU and NEWM are used for V_(H).For example, FR1-3 can come from EU and FR 4 from NEWM.

[0038] Other modifications, such as Asn-linked glycosylation sites, canbe introdueced into a chimerized, human, or humanized Immu31 antibody byconventional oligonucleotide directed site-specific mutagenesis.Detailed protocols for oligonucleotide-directed mutagenesis and relatedtechniques for mutagenesis of cloned DNA are well known. For example,see Sambrook et al. and Ausubel et al. above.

[0039] For example, to introduce an Asn in position 18 of hImmu31Vκ(FIG. 4B), one may alter codon 18 from AGG for Arg to AAC for Asn. Toaccomplish this, a single stranded DNA template containing the antibodylight chain sequence is prepared from a suitable strain of E. coli(e.g., dut⁻, ung⁻) in order to obtain a single strand DNA moleculecontaining a small number of uracils in place of thymidine. Such a DNAtemplate can be obtained by M13 cloning or by in vitro transcriptionusing a SP6 promoter. See, for example, Ausubel et al., eds., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, 1987. Anoligonucleotide containing the mutated sequence is synthesizedconventionally, annealed to the single-stranded template and the producttreated with T4 DNA polymerase and T4 DNA ligase to produce adouble-stranded DNA molecule. Transformation of wild type E. (dut⁺,ung⁺) cells with the double-stranded DNA provides an efficient recoveryof mutated DNA.

[0040] Alternatively, an Asn-linked glycosylation site can be introducedinto an antibody light chain using an oligonucleotide containing thedesired mutation as the primer and DNA clones of the variable regionsfor the Vk chain, or by using RNA from cells that produce the antibodyof interest as a template. Also see, Huse, in ANTIBODY ENGINEERING: APRACTICAL GUIDE, Boerrebaeck, ed., W. H. Freeman & Co., pp. 103-120,1992. Site-directed mutagenesis can be performed, for example, using theTRANSFORMER™ kit (Clonetech, Palo Alto, Calif.) according to themanufacturer's instructions.

[0041] Alternatively, a glycosylation site can be introduced bysynthesizing an antibody chain with mutually priming oligonucleotides,one such containing the desired mutation. See, for example, Uhlmann,Gene 71: 29 (1988); Wosnick et al., Gene 60: 115 (1988); Ausubel et al.,above, which are incorporated by reference.

[0042] Although the general description above referred to theintroduction of an Asn glycosylation site in position 18 of the lightchain of an antibody, it will occur to the skilled artisan that it ispossible to introduce Asn-linked glycosylation sites elsewhere in thelight chain, or even in the heavy chain variable region.

[0043] 4. Production of Antibody Fragments

[0044] Antibody fragments which recognize specific epitopes can begenerated by known techniques. The antibody fragments are antigenbinding portions of an antibody, such as F(ab′)₂, Fab′, Fab, Fv, sFv andthe like. Other antibody fragments include, but are not limited to: theF(ab)′2 fragments which can be produced by pepsin digestion of theantibody molecule and the Fab′ fragments, which can be generated byreducing disulfide bridges of the F(ab)′₂ fragments. Alternatively, Fab′expression expression libraries can be constructed (Huse et al., 1989,Science, 246:1274-1281) to allow rapid and easy identification ofmonoclonal Fab′ fragments with the desired specificity. The presentinvention encompasses antibodies and antibody fragments.

[0045] A single chain Fv molecule (scFv) comprises a VL domain and a VHdomain. The VL and VH domains associate to form a target binding site.These two domains are further covalently linked by a peptide linker (L).A scFv molecule is denoted as either VL-L-VH if the VL domain is theN-terminal part of the scFv molecule, or as VH-L-VL if the VH domain isthe N-terminal part of the scFv molecule. Methods for making scFvmolecules and designing suitable peptide linkers are described in U.S.Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow,“Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W.Walker, “Single Chain Antibody Variable Regions,” TIBTECH, Vol 9:132-137 (1991). These references are incorporated herein by reference.

[0046] To obtain high-affinity scFv, an scFv library with a largerepertoire can be constructed by isolating V-genes from non-immunizedhuman donors using PCR primers corresponding to all known V_(H), V_(κ)and V_(λ) gene families. See, e.g., Vaughn et al., Nat. Biotechnol., 14:309-314 (1996). Following amplification, the V_(κ) and V_(λ) pools arecombined to form one pool. These fragments are ligated into a phagemidvector. The scFv linker, (Gly-Gly-Gly-Gly- Ser)₃, is then ligated intothe phagemid upstream of the V_(L) fragment. The V_(H) and linker-V_(L)fragments are amplified and assembled on the J_(H) region. The resultingV_(H)-linker-V_(L) fragments are ligated into a phagemid vector. Thephagemid library can be panned using filters, as described above, orusing immunotubes (Nunc; Maxisorp). Similar results can be achieved byconstructing a combinatorial immunoglobulin library from lymphocytes orspleen cells of immunized rabbits and by expressing the scFv constructsin P. pastoris. See, e.g., Ridder et al., Biotechnology, 13: 255-260(1995). Additionally, following isolation of an appropriate scFv,antibody fragments with higher binding affinities and slowerdissociation rates can be obtained through affinity maturation processessuch as CDR3 mutagenesis and chain shuffling. See, e.g., Jackson et al.,Br. J Cancer, 78: 181-188(1998); Osbourn et al., Immunotechnology, 2:181-196 (1996).

[0047] An antibody fragment can be prepared by proteolytic hydrolysis ofthe full length antibody or by expression in E. coli or another host ofthe DNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 100 Kd fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfydryl groupsresulting from cleavage of disulfide linkages, to produce 50 Kd Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoffet al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

[0048] Another form of an antibody fragment is a peptide coding for asingle complementarity-determining region (CDR). A CDR is a segment ofthe variable region of an antibody that is complementary in structure tothe epitope to which the antibody binds and is more variable than therest of the variable region. Accordingly, a CDR is sometimes referred toas hypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

[0049] Other methods of cleaving antibodies, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments, or other enzymatic, chemical or genetic techniques mayalso be used, so long as the fragments bind to the antigen that isrecognized by the intact antibody.

[0050] 5. Fusion Proteins

[0051] The antibody fusion proteins of the present invention comprisetwo or more antibodies or fragments thereof and each of the antibodiesthat compose this fusion protein can contain a therapeutic agent ordiagnostic agent. In other words, the antibody fusion protein orfragment thereof can comprise at least one first anti-AFP MAb orfragment thereof and at least one second MAb or fragment thereof that isnot an anti-AFP MAb. In a preferred embodiement, the anti-AFP antibodyor fragment thereof is an Immu31 antibody or fragment thereof.Preferably, the second MAb is a carcinoma-associated antibody, such asan antibody against CEA, EGP-1, EGP-2 (e.g., 17-1A), MUC-1, MUC-2,MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3,Ep-CAM, Tn, and Thomson-Friedenreich antigens, tumor necrosis antigens,tenascin, an oncogene, an oncogene product, IL-6, IGF-1, IGFR-1, tumorangiogenesis antigens, such as vascular endothelium growth factor(VEGF), placental growth factor (PIGF), ED-B fibronectin, and othervascular growth factors, Ga 733, 17-1A, ferritin and acidic isoferritin(AIF) of primary hepatic carcinoma, or a combination thereof.

[0052] Additionally, one or more of the antibodies or fragments thereofthat comprise the antibody fusion protein can have at least onetherapeutic or diagnostic/detection agent attached. Further, thediagnostic/detection agents or therapeutic agents need not be the samebut can be different therapeutic agents; for example, one can attach adrug and a radioisotope to the same fusion protein. Particulary, an IgGcan be radiolabeled with ¹³¹I and attached to a drug. The ¹³¹I can beincorporated into the tyrosine of the IgG and the drug attached to theepsilon amino group of the IgG lysines. Both therapeutic and diagnosticagents also can be attached to reduced SH groups and to the carbohydrateside chains.

[0053] Also preferred, the antibody fusion protein of the presentinvention comprises at least two anti-AFP monoclonal antibodies orfragments thereof, and these may be to different epitopes of thealphafetoprotein antigen or of different human immunoglobulin backbonesequences (or IgGs). Preferably, the anti-AFP antibodies or fragmentsthere of are Immu31 antibodies or fragments thereof.

[0054] Multispecific and Multivalent Antibodies

[0055] In another embodiment of the instant invention is a conjugatedmultivalent ImMu31 antibody. Compositions and methods for multivalent,multispecific agents are described in Rossi et al., U.S. patentapplication Ser. No: 60/436,359, filed Dec. 24, 2002, and U.S. patentapplication Ser. No. 60/464,532, filed Apr. 23, 2003, which areincorporated herein by reference in its entirety.

[0056] The Immu31 antibodies and fragments thereof of the presentinvention, as well as other antibodies with different specificities foruse in combination therapy, can be made as a multispecific antibody,comprising at least one binding site to an alpha fetoprotein antigen andat least one binding site to another antigen, or a multivalent antibodycomprising multiple binding sites to the same epitope or antigen. In apreferred embodiment, the multispecific antibody or fragment thereofcomprises at least one binding site to an Immu31 epitope and at leastone binding site that is not to the AFP antigen. The Immu31 epitope isan epitope on the AFP antigen that is recognized by the Immu31antibodies of the present invention. Also preferred, the multispecificantibody or fragment thereof comprises at least one binding site to anImmu31 epitope and at least one binding site to a different epitope onthe AFP antigen.

[0057] The present invention provides a bispecific antibody or antibodyfragment having at least one binding region that specifically binds AFPand at least one other binding region that specifically binds anothertargeted cell marker or a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment. Preferably, the bispecific antibody bindsto an Immu31 epitope in the AFP antigen.

[0058] A variety of recombinant methods can be used to producebi-specific antibodies and antibody fragments. For example, bi-specificantibodies and antibody fragments can be produced in the milk oftransgenic livestock. See, e.g., Colman, A., Biochem. Soc. Symp., 63:141-147, 1998; U.S. Pat. No. 5,827,690. Two DNA constructs are preparedwhich contain, respectively, DNA segments encoding paired immunoglobulinheavy and light chains. The fragments are cloned into expression vectorswhich contain a promoter sequence that is preferentially expressed inmammary epithelial cells. Examples include, but are not limited to,promoters from rabbit, cow and sheep casein genes, the cowα-lactoglobulin gene, the sheep β-lactoglobulin gene and the mouse wheyacid protein gene. Preferably, the inserted fragment is flanked on its3′ side by cognate genomic sequences from a mammary-specific gene. Thisprovides a polyadenylation site and transcript-stabilizing sequences.The expression cassettes are coinjected into the pronuclei offertilized, mammalian eggs, which are then implanted into the uterus ofa recipient female and allowed to gestate. After birth, the progeny arescreened for the presence of both transgenes by Southern analysis. Inorder for the antibody to be present, both heavy and light chain genesmust be expressed concurrently in the same cell. Milk from transgenicfemales is analyzed for the presence and functionality of the antibodyor antibody fragment using standard immunological methods known in theart. The antibody can be purified from the milk using standard methodsknown in the art.

[0059] Other recent methods for producing bsAbs include engineeredrecombinant Abs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bi-specific fusion proteins canbe produced using molecular engineering. In one form, the bi-specificfusion protein is monovalent, consisting of, for example, a scFv with asingle binding site for one antigen and a Fab fragment with a singlebinding site for a second antigen. In another form, the bi-specificfusion protein is divalent, consisting of, for example, an IgG with twobinding sites for one antigen and two scFv with two binding sites for asecond antigen.

[0060] An anti-AFP multivalent antibody or fragment thereof is alsocontemplated in the present invention. Preferably, the anti-AFPmultivalent antibody or fragment thereof is an Immu31 multivalentantibody or fragment thereof. This multivalent antibody is constructedby association of a first and a second polypeptide. The firstpolypeptide comprises a first single chain Fv molecule covalently linkedto a first immunoglobulin-like domain which preferably is animmunoglobulin light chain variable region domain. The secondpolypeptide comprises a second single chain Fv molecule covalentlylinked to a second immunoglobulin-like domain which preferably is animmunoglobulin heavy chain variable region domain. Each of the first andsecond single chain Fv molecules forms a target binding site, and thefirst and second immunoglobulin-like domains associate to form a thirdtarget binding site.

[0061] A single chain Fv molecule with the VL-L-VH configuration,wherein L is a linker, may associate with another single chain Fvmolecule with the VH-L-VL configuration to form a bivalent dimer. Inthis case, the VL domain of the first scFv and the VH domain of thesecond scFv molecule associate to form one target binding site, whilethe VH domain of the first scFv and the VL domain of the second scFvassociate to form the other target binding site.

[0062] Another embodiment of the present invention is an Immu31bispecific, trivalent antibody comprising two heterologous polypeptidechains associated non-covalently to form three binding sites, two ofwhich have affinity for one target and a third which has affinity for ahapten that can be made and attached to a carrier for a diagnosticand/or therapeutic agent. Preferably, the antibody has two Immu31binding sites and one CEA or MUC1 binding site. The bispecific,trivalent targeting agents have two different scFvs, one scFv containstwo V_(H) domains from one antibody connected by a short linker to theV_(L) domain of another antibody and the second scFv contains two V_(L)domains from the first antibody connected by a short linker to the V_(H)domain of the other antibody. The methods for generating multivalent,multispecific agents from V_(H) and V_(L) domains provide thatindividual chains synthesized from a DNA plasmid in a host organism arecomposed entirely of V_(H) domains (the V_(H)-chain) or entirely ofV_(L) domains (the V_(L)-chain) in such a way that any agent ofmultivalency and multispecificity can be produced by non-covalentassociation of one V_(H)-chain with one V_(L)-chain. For example,forming a trivalent, trispecific agent, the V_(H)-chain will consist ofthe amino acid sequences of three V_(H) domains, each from an antibodyof different specificity, joined by peptide linkers of variable lengths,and the V_(L)-chain will consist of complementary V_(L) domains, joinedby peptide linkers similar to those used for the V_(H)-chain. Since theV_(H) and V_(L) domains of antibodies associate in an anti-parallelfashion, the preferred method in this invention has the V_(L) domains inthe V_(L)-chain arranged in the reverse order of the V_(H) domains inthe V_(H)-chain.

[0063] Diabodies, Triabodies and Tetrabodies

[0064] The anti-AFP antibodies and fragments thereof of the presentinvention can also be used to prepare functional bispecific single-chainantibodies (bscAb), also called diabodies, and can be produced inmammalian cells using recombinant methods. Preferably, the anti-AFPantibody or fragment thereof is an Immu31 antibody or fragment thereof.See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995,incorporated. For example, bscAb are produced by joining twosingle-chain Fv fragments via a glycine-serine linker using recombinantmethods. The V light-chain (V_(L)) and V heavy-chain (V_(H)) domains oftwo antibodies of interest are isolated using standard PCR methods. TheV_(L) and V_(H) cDNA's obtained from each hybridoma are then joined toform a single-chain fragment in a two-step fusion PCR. The first PCRstep introduces the (Gly₄-Ser₁)₃ linker, and the second step joins theV_(L) and V_(H) amplicons. Each single chain molecule is then clonedinto a bacterial expression vector. Following amplification, one of thesingle-chain molecules is excised and sub-cloned into the other vector,containing the second single-chain molecule of interest. The resultingbscAb fragment is subcloned into an eukaryotic expression vector.Functional protein expression can be obtained by transfecting the vectorinto chinese hamster ovary cells. Bispecific fusion proteins areprepared in a similar manner. Bispecific single-chain antibodies andbispecific fusion proteins are included within the scope of the presentinvention.

[0065] For example, a humanized, chimeric or human or murine Immu31monoclonal antibody can be used to produce antigen specific diabodies,triabodies, and tetrabodies. The monospecific diabodies, triabodies, andtetrabodies bind selectively to targeted antigens and as the number ofbinding sites on the molecule increases, the affinity for the targetcell increases and a longer residence time is observed at the desiredlocation. For diabodies, the two chains comprising the V_(H) polypeptideof the humanized Immu31 MAb connected to the V_(K) polypeptide of thehumanized Immu31 MAb by a five amino acid residue linker are utilized.Each chain forms one half of the humanized Immu31 diabody. In the caseof triabodies, the three chains comprising V_(H) polypeptide of thehumanized Immu31 MAb connected to the V_(K) polypeptide of the humanizedImmu31 MAb by no linker are utilized. Each chain forms one third of thehImmu31 triabody.

[0066] Also contemplated in the present invention is a bi-specificantibody or antibody fragment having at least one arm that is reactiveagainst a targeted tissue such as AFP and at least one other arm that isreactive against a targetable construct. Preferably, one arm of thebispecific antibody binds the Immu 31 epitope. The targetable constructis comprised of a carrier portion and at least 2 units of a recognizablehapten. Examples of recognizable haptens include, but are not limitedto, histamine succinyl glycine (HSG) and fluorescein isothiocyanate. Thetargetable construct may be conjugated to a variety of agents useful fortreating or identifying diseased tissue. The targetable construct can beof diverse structure, but is selected not only to avoid eliciting animmune responses, but also for rapid in vivo clearance when used withinthe bsAb targeting method. Hydrophobic agents are best at elicitingstrong immune responses, whereas hydrophilic agents are preferred forrapid in vivo clearance; thus, a balance between hydrophobic andhydrophilic needs to be established. This is accomplished, in part, byrelying on the use of hydrophilic chelating agents to offset theinherent hydrophobicity of many organic moieties. Also, subunits of thetargetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic. Aside frompeptides, carbohydrates may be used.

[0067] Large quantities of bscAb and fusion proteins can be producedusing Escherichia coil expression systems. See, e.g., Zhenping et al.,Biotechnology, 14: 192-196, 1996. A functional bscAb can be produced bythe coexpression in E. coli of two “cross-over” scFv fragments in whichthe V_(L) and V_(H) domains for the two fragments are present ondifferent polypeptide chains. The V light-chain (V_(L)) and Vheavy-chain (V_(H)) domains of two antibodies of interest are isolatedusing standard PCR methods. The cDNA's are then ligated into a bacterialexpression vector such that C-terminus of the V_(L) domain of the firstantibody of interest is ligated via a linker to the N-terminus of theV_(H) domain of the second antibody. Similarly, the C-terminus of theV_(L) domain of the second antibody of interest is ligated via a linkerto the N-terminus of the V_(H) domain of the first antibody. Theresulting dicistronic operon is placed under transcriptional control ofa strong promoter, e.g., the E. coli alkaline phosphatase promoter whichis inducible by phosphate starvation. Alternatively, single-chain fusionconstructs have successfully been expressed in E. coli using the lacpromoter and a medium consisting of 2% glycine and 1% Triton X-100. See,e.g., Yang et al., Appl. Environ. Microbiol., 64: 2869-2874, 1998. An E.coli, heat-stable, enterotoxin II signal sequence is used to direct thepeptides to the periplasmic space. After secretion, the two peptidechains associate to form a non-covalent heterodimer which possesses bothantigen binding specificities. The bscAb is purified using standardprocedures known in the art, e.g., Staphylococcal protein Achromatography.

[0068] Functional bscAbs and fusion proteins also can be produced in themilk of transgenic livestock. See, e.g., Colman, A., Biochem. Soc.Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690. The bscAb fragment,obtained as described above, is cloned into an expression vectorcontaining a promoter sequence that is preferentially expressed inmammary epithelial cells. Examples include, but are not limited to,promoters from rabbit, cow and sheep casein genes, the cowα-lactoglobulin gene, the sheep β-lactoglobulin gene and the mouse wheyacid protein gene. Preferably, the inserted bscAb is flanked on its 3′side by cognate genomic sequences from a mammary-specific gene. Thisprovides a polyadenylation site and transcript-stabilizing sequences.The expression cassette is then injected into the pronuclei offertilized, mammalian eggs, which are then implanted into the uterus ofa recipient female and allowed to gestate. After birth, the progeny arescreened for the presence of the introduced DNA by Southern analysis.Milk from transgenic females is analyzed for the presence andfunctionality of the bscAb using standard immunological methods known inthe art. The bscAb can be purified from the milk using standard methodsknown in the art. Transgenic production of bscAb in milk provides anefficient method for obtaining large quantities of bscAb.

[0069] Functional bscAb and fusion proteins also can be produced intransgenic plants. See, e.g., Fiedler et al., Biotech., 13: 1090-1093,1995; Fiedler et al., Immunotechnology, 3: 205-216, 1997. Suchproduction offers several advantages including low cost, large scaleoutput and stable, long term storage. The bscAb fragment, obtained asdescribed above, is cloned into an expression vector containing apromoter sequence and encoding a signal peptide sequence, to direct theprotein to the endoplasmic recticulum. A variety of promoters can beutilized, allowing the practitioner to direct the expression product toparticular locations within the plant. For example, ubiquitousexpression in tobacco plants can be achieved by using the strongcauliflower mosaic virus 35S promoter, while organ specific expressionis achieved via the seed specific legumin B4 promoter. The expressioncassette is transformed according to standard methods known in the art.Transformation is verified by Southern analysis. Transgenic plants areanalyzed for the presence and functionality of the bscAb using standardimmunological methods known in the art. The bscAb can be purified fromthe plant tissues using standard methods known in the art.

[0070] Additionally, transgenic plants facilitate long term storage ofbscAb and fusion proteins. Functionally active scFv proteins have beenextracted from tobacco leaves after a week of storage at roomtemperature. Similarly, transgenic tobacco seeds stored for 1 year atroom temperature show no loss of scFv protein or its antigen bindingactivity.

[0071] Functional bscAb and fusion proteins also can be produced ininsect cells. See, e.g., Mahiouz et al., J Immunol. Methods, 212:149-160 (1998). Insect-based expression systems provide a means ofproducing large quantities of homogenous and properly folded bscAb. Thebaculovirus is a widely used expression vector for insect cells and hasbeen successfully applied to recombinant antibody molecules. See, e.g.,Miller, L. K., Ann. Rev. Microbiol., 42: 177 (1988); Bei et al., J.Immunol. Methods, 186: 245 (1995). Alternatively, an inducibleexpression system can be utilized by generating a stable insect cellline containing the bscAb construct under the transcriptional control ofan inducible promoter. See, e.g., Mahiouz et al., J. Immunol. Methods,212: 149-160 (1998). The bscAb fragment, obtained as described above, iscloned into an expression vector containing the Drosphilametallothionein promoter and the human HLA-A2 leader sequence. Theconstruct is then transfected into D. melanogaster SC-2 cells.Expression is induced by exposing the cells to elevated amounts ofcopper, zinc or cadmium. The presence and functionality of the bscAb isdetermined using standard immunological methods known in the art.Purified bscAb is obtained using standard methods known in the art.

[0072] The ultimate use of the bispecific diabodies described herein isfor pre-targeting Immu31 positive tumors for subsequent specificdelivery of diagnostic/detection or therapeutic agents. These diabodiesbind selectively to targeted antigens allowing for increased affinityand a longer residence time at the desired location. Moreover,non-antigen bound diabodies are cleared from the body quickly andexposure of normal tissues is minimized. The diagnostic/detection andtherapeutic agents can include isotopes, drugs, toxins, cytokines,hormones, growth factors, conjugates, radionuclides, and metals. Forexample, gadolinium metal is used for magnetic resonance imaging (MRI).Examples of radionuclides are ²²⁵Ac, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In,¹³¹I, ¹²⁵I, ¹²³I, ^(99m)Tc, ^(94m)Tc, ⁸⁶Re, ¹⁸⁸Re, ¹⁷⁷Lu, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ²¹²Bi, ²¹³Bi, ³²P, ¹¹C, ¹³N, 15O, ⁷⁶Br, and ²¹¹At. Otherradionuclides are also available as diagnostic and therapeutic agents,especially those in the energy range of 60 to 4,000 keV.

[0073] More recently, a tetravalent tandem diabody (termed tandab) withdual specificity has also been reported (Cochlovius et al., CancerResearch (2000) 60: 4336-4341). The bispecific tandab is a dimer of twoidentical polypeptides, each containing four variable domains of twodifferent antibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in anorientation to facilitate the formation of two potential binding sitesfor each of the two different specificities upon self-association.

[0074] 7. Immu31Immunoconjugates

[0075] Any of the anti-AFP antibodies or fragments thereof, or antibodyfusion proteins or fragments thereof of the present invention can beconjugated with one or more therapeutic and/or diagnostic/detectionagents. Generally, one therapeutic or diagnostic/detection agent isattached to each antibody or antibody fragment but more than onetherapeutic agent or diagnostic agent can be attached to the sameantibody, fusion protein, or fragment thereof. Such a therapeutic ordiagnostic/detection agent may be a peptide which bears adiagnostic/detection or therapeutic agent. An immunoconjugate retainsthe immunoreactivity of the antibody component, i.e., the antibodymoiety has about the same or slightly reduced ability to bind thecognate antigen after conjugation as before conjugation.

[0076] A wide variety of diagnostic/detection and therapeutic agents canbe advantageously conjugated to the antibody, fusion protein, orfragment thereof of the present invention. In a preferred embodiment,the diagnostic/detection agents are selected from the group consistingof radioisotopes for nuclear imaging, intraoperative and endoscopicdetection, enhancing agents for use in magnetic resonance imaging or inultrasonography, radiopaque and contrast agents for X-rays and computedtomography, and fluorescent compounds for fluoroscopy, includingendoscopic fluoroscopy. Fluorescent and radioactive agents conjugated toantibodies or used in bispecific, pretargeting methods, are particularlyuseful for endoscopic, intraoperative or intravascular detection of thetargeted antigens associated with diseased tissues or clusters of cells,such as malignant tumors, as disclosed in Goldenberg U.S. Pat. Nos.5,716,595, 6, 096,289 and U.S. application Ser. No. 09/348,818,incorporated herein by reference in their entirety, particularly withgamma-, beta-, and positron-emitters. Radionuclides useful for positronemission tomography include, but are not limited to: F-18, Mn-51,Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m,Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120, and I-124.

[0077] The therapeutic agents recited here are those agents that alsoare useful for administration separately with a naked antibody, asdescribed herein. Therapeutic agents include, for example,chemotherapeutic drugs such as vinca alkaloids and other alkaloids,anthracyclines, epidophyllotoxins, taxanes, antimetabolites, alkylatingagents, antibiotics, COX-2 inhibitors, antimitotics, antiangiogenic andapoptotoic agents, particularly doxorubicin, methotrexate, taxol,CPT-11, camptothecans, and others from these and other classes ofanticancer agents, and the like. Other useful cancer chemotherapeuticdrugs for the preparation of immunoconjugates and antibody fusionproteins include nitrogen mustards, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs,purine analogs, platinum coordination complexes, hormones, toxins (e.g.,RNAse, Psudomonas exotoxin), and the like. Suitable chemotherapeuticagents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.1985), as well as revised editions of these publications. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

[0078] A toxin, such as Pseudomonas exotoxin, may also be complexed toor form the therapeutic agent portion of an immunoconjugate of theImmu31 antibody or fragment thereof of the present invention.Additionally, the toxin may be used in combination with a naked Immu31antibody or fragment thereof, an Immu31 fusion protein or fragmentthereof, or a Immu31 antibody or fragment thereof conjugated to adifferent therapeutic agent. Other toxins suitably employed in thepreparation of such conjugates or other fusion proteins, include ricin,abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin. See, for example, Pastan et al.,Cell 47:641 (1986), and Goldenberg, CA—A Cancer Journal for Clinicians44:43 (1994). Additional toxins suitable for use in the presentinvention are known to those of skill in the art and are disclosed inU.S. Pat. No. 6,077,499, which is incorporated in its entirety byreference. These can be derived, for example, from animal, plant andmicrobial sources, or chemically or recombinantly engineered. The toxincan be a plant, microbial, or animal toxin, or a synthetic variationthereof.

[0079] An immunomodulator, such as a cytokine may also be conjugated to,or form the therapeutic agent portion of the Immu31 immunoconjugate, orbe administered unconjugated to the chimeric, humanized or humananti-AFP antibody, fusion protein, or fragment thereof of the presentinvention. As used herein, the term “immunomodulator” includescytokines, stem cell growth factors, lymphotoxins, such as tumornecrosis factor (TNF), and hematopoietic factors, such as interleukins(e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12 and IL-18),colony stimulating factors (e.g., granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)),interferons (e.g., interferons-α, -β and -γ), the stem cell growthfactor designated “S1 factor,” erythropoietin and thrombopoietin.Examples of suitable immunomodulator moieties include IL-2, IL-6, IL-10,IL-12, IL-18, interferon-γ, TNF-α, and the like. Alternatively, subjectscan receive a naked Immu31 antibody or fragment thereof, or naked fusionprotein or fragment thereof, and a separately administered cytokine,which can be administered before, concurrently or after administrationof the naked Immu31 antibody or fragment, or naked Immu31 fusion proteinor fragment thereof. The Immu31 antibody or fragment there or fusionprotein or fragment thereof of may also be conjugated to animmunomodulator. The immunomodulator may also be conjugated to a hybridantibody consisting of one or more antibodies or antibody fragmentsbinding to different antigens. Such an antigen may also be animmunomodulator. For example, CD40 or other immunomodulators may beadministered in combination with a Immu31 antibody or fragment thereofeither together, before or after the antibody combinations areadministered.

[0080] Furthermore, an Immu31 antibody or fragment thereof, or fusionprotein or fragment thereof may comprise a γ-emitting radionuclide or apositron-emitter useful for diagnostic imaging. Examples ofdiagnostic/detection agents include diverse labels, radionuclides,chelators, dyes, contrast agents, fluorescent compounds, chromagens, andother marker moieties. Radionuclides useful for positron emissiontomography include, but are not limited to: ¹⁸F, ⁵¹Mn, ^(52m)Mn, ⁵²Fe,⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁸Ga, ⁷² As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb ⁸³Sr, ⁸⁶Y, ⁸⁹Zr,^(94m)Tc, ¹¹⁰In, ¹²⁰I, and ¹²⁴I. Total decay energies of usefulpositron-emitting radionuclides are preferably<2,000 keV, morepreferably under 1,000 keV, and most preferably<700 keV. Radionuclidesuseful as diagnostic agents utilizing gamma-ray detection include, butare not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75,Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197, andT1-201. Decay energies of useful gamma-ray emitting radionuclides arepreferably 20-2000 keV, more preferably 60-600 keV, and most preferably100-300 keV.

[0081] Additionally, radionuclides suitable for treating a diseasedtissue include, but are not limited to, P-32, P-33, Sc-47, Fe-59, Cu-64,Cu-67, Se-75, As-77, Sr-89, Y-90, Mo-99, Rh-105, Pd-109, Ag-111, I-125,I-131, Pr-142, Pr-143, Pm-149, Sm-153, Tb-161, Ho-166, Er-169, Lu-177,Re-186, Re-188, Re-189, Ir-194, Au-198, Au-199, Pb-211, Pb-212, andBi-213, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161, Os-189m, Ir-192, Dy-152, At-211, Bi-212, Ra-223, Rn-219,Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.

[0082] Suitable diagnostic imaging isotopes are usually in the range of20 to 2,000 keV, while suitable therapeutic radionuclides are usually inthe range of 20 to 10,000 keV. See for example, U.S. patent applicationentitled “Labeling Targeting Agents with Gallium-68”-Inventors G. L.Griffiths and W. J. McBride, (U.S. Provisional Application No.60/342,104), which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga,^(94m)Tc. and the like, for imaging purposes and which is incorporatedin its entirety by reference. A suitable radionuclide is an Augeremitter, and preferably has an energy of less than 1000 keV. Alsopreferred is a P emitter and has an energy between 20 and 5000 keV or ana emitter and has an energy between 2000 and 10,000 keV.

[0083] A therapeutic or diagnostic/detection agent can be attached atthe hinge region of a reduced antibody component via disulfide bondformation. As an alternative, such peptides can be attached to theantibody component using a heterobifunctional cross-linker, such asN-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J.Cancer 56: 244 (1994). General techniques for such conjugation are wellknown in the art. See, for example, Wong, CHEMISTRY OF PROTEINCONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et al.,“Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same peptide that is bound to athiol group, or the carbohydrate moiety can be used to bind a differentpeptide.

[0084] Methods for conjugating peptides to antibody components via anantibody carbohydrate moiety are well known to those of skill in theart. See, for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shihet al., Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

[0085] However, if the Fc region is absent, for example, if the antibodyused as the antibody component of the immunoconjugate is an antibodyfragment, it is still possible to attach a diagnostic/detection atherapeutic agent. A carbohydrate moiety can be introduced into thelight chain variable region of a full-length antibody or antibodyfragment. See, for example, Leung et al., J. Immunol. 154: 5919 (1995);Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat.No. 6,254,868, all of which are incoporated in their entirety byreference. The engineered carbohydrate moiety is used to attach thetherapeutic or diagnostic agent.

[0086] Targetable Constructs

[0087] The targetable construct can be of diverse structure, but isselected not only to avoid eliciting an immune responses, but also forrapid in vivo clearance when used within the bsAb targeting method.Hydrophobic agents are best at eliciting strong immune responses,whereas hydrophilic agents are preferred for rapid in vivo clearance;thus, a balance between hydrophobic and hydrophilic needs to beestablished. This is accomplished, in part, by relying on the use ofhydrophilic chelating agents to offset the inherent hydrophobicity ofmany organic moieties. Also, subunits of the targetable construct may bechosen which have opposite solution properties, for example, peptides,which contain amino acids, some of which are hydrophobic and some ofwhich are hydrophilic. Aside from peptides, carbohydrates may be used.

[0088] Peptides having as few as two amino-acid residues may be used,preferably two to ten residues, if also coupled to other moieties suchas chelating agents. The linker should be a low molecular weightconjugate, preferably having a molecular weight of less than 50,000daltons, and advantageously less than about 20,000 daltons, 10,000daltons or 5,000 daltons, including the metal ions in the chelates. Forinstance, the known peptide DTPA-Tyr-Lys(DTPA)-OH (wherein DTPA isdiethylenetriaminepentaacetic acid) has been used to generate antibodiesagainst the indium-DTPA portion of the molecule. However, by use of thenon-indium-containing molecule, and appropriate screening steps, new Absagainst the tyrosyl-lysine dipeptide can be made. More usually, theantigenic peptide will have four or more residues, such as the peptideDOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)—NH₂, wherein DOTA is1,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the histaminesuccinyl glycyl group of the formula:

[0089] The non-metal-containing peptide may be used as an immunogen,with resultant Abs screened for reactivity against the Phe-Lys-Tyr-Lysbackbone.

[0090] The invention also contemplates the incorporation of unnaturalamino acids, e.g., D-amino acids, into the backbone structure to ensurethat, when used with the final bsAb/linker system, the arm of the bsAbwhich recognizes the linker moiety is completely specific. The inventionfurther contemplates other backbone structures such as those constructedfrom non-natural amino acids and peptoids.

[0091] The peptides to be used as immunogens are synthesizedconveniently on an automated peptide synthesizer using a solid-phasesupport and standard techniques of repetitive orthogonal deprotectionand coupling. Free amino groups in the peptide, that are to be usedlater for chelate conjugation, are advantageously blocked with standardprotecting groups such as an acetyl group. Such protecting groups willbe known to the skilled artisan. See Greene and Wuts Protective Groupsin Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). When thepeptides are prepared for later use within the bsAb system, they areadvantageously cleaved from the resins to generate the correspondingC-terminal amides, in order to inhibit in vivo carboxypeptidaseactivity.

[0092] The haptens of the immunogen comprise an immunogenic recognitionmoiety, for example, a chemical hapten. Using a chemical hapten,preferably the HSG hapten, high specificity of the linker for theantibody is exhibited. This occurs because antibodies raised to the HSGhapten are known and can be easily incorporated into the appropriatebispecific antibody. Thus, binding of the linker with the attachedhapten would be highly specific for the antibody or antibody fragment.

[0093] Chelate Moieties

[0094] The presence of hydrophilic chelate moieties on the linkermoieties helps to ensure rapid in vivo clearance. In addition tohydrophilicity, chelators are chosen for their metal-binding properties,and are changed at will since, at least for those linkers whose bsAbepitope is part of the peptide or is a non-chelate chemical hapten,recognition of the metal-chelate complex is no longer an issue.

[0095] A chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated. For example, a therapeutically useful immunoconjugate can beobtained by conjugating a photoactive agent or dye to an antibody fusionprotein. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been termed photoradiation,phototherapy, or photodynamic therapy (Jori et al. (eds.), PHOTODYNAMICTHERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);idem., Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin.Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422(1989); Pelegrin et al., Cancer 67:2529 (1991). However, these earlierstudies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

[0096] Particularly useful metal-chelate combinations include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with ⁴⁷SC,⁵²Fe, ⁵⁵Co, ⁶⁷Ga, ⁶⁸ Ga, ¹²¹¹In, ⁸⁹Zr, ⁹⁰Y, ¹⁶¹Tb, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi,and ²²⁵Ac for radio-imaging and RAIT. The same chelators, when complexedwith non-radioactive metals, such as Mn, Fe and Gd can be used for MRI,when used along with the bsAbs of the invention. Macrocyclic chelatorssuch as NOTA (1,4,7-triaza-cyclononane-N,N′,N″-triacetic acid), DOTA,and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) areof use with a variety of metals and radiometals, most particularly withradionuclides of Ga, Y and Cu, respectively.

[0097] DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group IIa andGroup IIIa metal cations. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelators such as macrocyclic polyethers are of interest forstably binding nuclides such as ²²³Ra for RAIT. Porphyrin chelators maybe used with numerous radiometals, and are also useful as certain coldmetal complexes for bsAb-directed immuno-phototherapy. More than onetype of chelator may be conjugated to a carrier to bind multiple metalions, e.g., cold ions, diagnostic radionuclides and/or therapeuticradionuclides. Particularly useful therapeutic radionuclides include,but are not limited to, ³²P, ³³P, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag,¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra and ²²⁵Ac. Particularlyuseful diagnostic/detection radionuclides include, but are not limitedto, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸ Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc,^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd and ¹⁷⁵Lu.

[0098] Chelators such as those disclosed in U.S. Pat. No. 5,753,206,especially thiosemi-carbazonylglyoxylcysteine (Tscg-Cys) andthiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators areadvantageously used to bind soft acid cations of Tc, Re, Bi and othertransition metals, lanthanides and actinides that are tightly bound tosoft base ligands, especially sulfur- or phosphorus-containing ligands.It can be useful to link more than one type of chelator to a peptide,e.g., a DTPA or similar chelator for, say In(III) cations, and athiol-containing chelator, e.g., Tscg-Cys, for Tc cations. Becauseantibodies to a di-DTPA hapten are known (Barbet '395, supra) and arereadily coupled to a targeting antibody to form a bsAb, it is possibleto use a peptide hapten with cold diDTPA chelator and another chelatorfor binding a radioisotope, in a pretargeting protocol, for targetingthe radioisotope. One example of such a peptide isAc-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys-)—NH₂. This peptide can bepreloaded with In(III) and then labeled with 99-m-Tc cations, theIn(III) ions being preferentially chelated by the DTPA and the Tccations binding preferentially to the thiol-containing Tscg-Cys. Otherhard acid chelators such as NOTA, DOTA, TETA and the like can besubstituted for the DTPA groups, and Mabs specific to them can beproduced using analogous techniques to those used to generate theanti-di-DTPA Mab.

[0099] It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the linker, e.g., with differentchelate ring sizes, to bind preferentially to two different hard acid orsoft acid cations, due to the differing sizes of the cations, thegeometries of the chelate rings and the preferred complex ion structuresof the cations. This will permit two different metals, one or both ofwhich may be radioactive or useful for MRI enhancement, to beincorporated into a linker for eventual capture by a pretargeted bsAb.

[0100] Preferred chelators include NOTA, DOTA and Tscg and combinationsthereof. These chelators have been incorporated into a chelator-peptideconjugate motif as exemplified in the following constructs:

[0101] (a) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)—NH₂;

[0102] (b) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)—NH₂;

[0103] (c) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)—NH₂;

[0104] The chelator-peptide conjugates (d) and (e), above, has beenshown to bind ⁶⁸Ga and is thus useful in positron emission tomography(PET) applications.

[0105] Chelators are coupled to the linker moieties using standardchemistries which are discussed more fully in the working Examplesbelow. Briefly, the synthesis of the peptideAc-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)—NH₂ was accomplished by firstattaching Aloc-Lys(Fmoc)-OH to a Rink amide resin on the peptidesynthesizer. The protecting group abbreviations “Aloc” and “Fmoc” usedherein refer to the groups allyloxycarbonyl and fluorenylmethyloxycarbonyl. The Fmoc-Cys(Trt)-OH and TscG were then added to the sidechain of the lysine using standard Fmoc automated synthesis protocols toform the following peptide: Aloc-Lys(Tscg-Cys(Trt)-rink resin. The Alocgroup was then removed. The peptide synthesis was then continued on thesynthesizer to make the following peptide:(Lys(Aloc)-D-Tyr-Lys(Aloc)-Lys(Tscg-Cys(Trt)-)-rink resin. FollowingN-terminus acylation, and removal of the side chain Aloc protectinggroups. The resulting peptide was then treated with activatedN-trityl-HSG-OH until the resin gave a negative test for amines usingthe Kaiser test. See Karacay et al. Bioconjugate Chem. 11:842-854(2000). The synthesis of Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys-)—NH₂,as well as the syntheses of DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)—NH₂; andDOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)—NH₂ are described in greater detailbelow.

[0106] Preparation of Metal Chelates

[0107] Chelator-peptide conjugates may be stored for long periods assolids. They may be metered into unit doses for metal-binding reactions,and stored as unit doses either as solids, aqueous or semi-aqueoussolutions, frozen solutions or lyophilized preparations. They may belabeled by well-known procedures. Typically, a hard acid cation isintroduced as a solution of a convenient salt, and is taken up by thehard acid chelator and possibly by the soft acid chelator. However,later addition of soft acid cations leads to binding thereof by the softacid chelator, displacing any hard acid cations which may be chelatedtherein. For example, even in the presence of an excess of cold¹¹¹InCl₃, labeling with 99m-Tc(V) glucoheptonate or with Tc cationsgenerated in situ with stannous chloride and Na99m-TcO₄ proceedsquantitatively on the soft acid chelator. Other soft acid cations suchas ¹⁸⁶Re, ¹⁸⁸Re, ²¹³Bi and divalent or trivalent cations of Mn, Co, Ni,Pb, Cu, Cd, Au, Fe, Ag (monovalent), Zn and Hg, especially ⁶⁴Cu and⁶⁷Cu, and the like, some of which are useful for radioimmunodiagnosis orradioimmunotherapy, can be loaded onto the linker peptide by analogousmethods. Re cations also can be generated in situ from perrhenate andstannous ions or a prereduced rhenium glucoheptonate or othertranschelator can be used. Because reduction of perrhenate requires morestannous ion (typically above 200 μg/mL final concentration) than isneeded for the reduction of Tc, extra care needs to be taken to ensurethat the higher levels of stannous ion do not reduce sensitive disulfidebonds such as those present in disulfide-cyclized peptides. Duringradiolabeling with rhenium, similar procedures are used as are used withthe Tc-99m. A preferred method for the preparation of ReO metalcomplexes of the Tscg-Cys- ligands is by reacting the peptide withReOCl₃(P(Ph₃)₂ but it is also possible to use other reduced species suchas ReO(ethylenediamine)₂.

[0108] 8. Humanized, Chimeric and Human Antibodies Use for Treatment andDiagnosis

[0109] Contemplated in the present invention is the use of murine,humanized, chimeric and human anti-AFP antibodies and fragments thereofin delivery methods of therapeutic and diagnostic/detection agents, andtherapeutic and diagnostic/detection methods. Preferably, the murine,chimeric, humanized and human anti-AFP antibodies and fragments thereofare chimeric, humanized or human Immu31 antibodies.

[0110] For example, a method of delivering a diagnostic/detection agent,a therapeutic agent, or a combintion thereof to a target comprising (i)administering to a subject the antibody or fragment thereof an antibody,fusion protein, or fragment thereof; (ii) waiting a sufficient amount oftime for an amount of the non-binding protein to clear the subject'sblood stream; and (iii) administering to said subject a carrier moleculecomprising a diagnostic/detection agent, a therapeutic agent, or acombination thereof, that binds to a binding site of said antibody.Preferably, the carrier molecule binds to more than one binding site ofthe antibody.

[0111] The present invention also contemplates methods of diagnosing ordetecting a malignancy in a subject. Diagnosis/detection may beaccomplished by administering a diagnostically effective amount of adiagnostic/detection immunoconjugate, comprising an anti-AFP monoclonalantibody or fragment thereof or a fusion protein or fragment thereof,wherein said anti-AFP MAb or fragment thereof or fuision protein orfragment thereof is bound to at least one diagnostic/detection agent,formulated in a pharmaceutically acceptable excipient, and detectingsaid label. Preferably, the anti-AFP antibody, fusion protein, orfragment thereof is an Immu31 antibody.

[0112] In a related vein, a method of diagnosing or detecting amalignancy in a subject comprising (i) performing an in vitro diagnosisassay on a specimen from said subject with a composition comprising aanti-AFP MAb or fragment thereof or a antibody fusion protein orfragment thereof of any one of the antibodies, fusion proteins, orfragments thereof of the present invention, is also considered.Preferably, the in vitro diagnosis assay is selected from the groupconsisting of immunoassays, RT-PCR and immunohistochemistry.

[0113] In the methods described herein, radioactive and non-radioactiveagents can be used as diagnostic agents. A suitable non-radioactivediagnostic agent is a contrast agent suitable for magnetic resonanceimaging, a radiopaque compound for X-rays or computed tomography, or acontrast agent suitable for ultrasound. Magnetic imaging agents include,for example, non-radioactive metals, such as manganese, iron andgadolinium, complexed with metal-chelate combinations that include2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, when used alongwith the antibodies of the invention. See U.S. Ser. No. 09/921,290 filedon Oct. 10, 2001, which is incorporated in its entirety by reference. Ina preferred embodiment, the contrast agent is an ultrasound-enhancingagent. Still preferred, the ultrasound-enhancing agent is a liposome.Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

[0114] Also described in the present invention is the use of murine,chimeric, humanized and human anti-AFP antibodies and fragments thereofin methods for treating malignancies. For example, a malignancy ofparticular interest in this patent is a cancer of the liver.Occasionally, ovarian carcinoma, and rarely gastrointestinal and lungcancers may produce AFP. Preferably, the anti-AFP antibodies andfragements thereof are Immu31 antibodies and fragments thereof. Themethod comprises administering to a subject a therapeutically effectiveamount of an antibody or fragment thereof or an antibody fusion proteinor fragment thereof comprising at least two MAbs or fragments thereof,wherein at least one anti-AFP MAb or fragment thereof or fusion proteinsor fragments thereof are any one of the antibodies of the presentinvention, formulated in a pharmaceutically suitable excipient. Inanother embodiment, a second MAb, fusion protein or fragment thereof isnot an anti-AFP antibody, fusion protein or fragment thereof.

[0115] In a related vein, a method of treating a cancer cell in asubject comprising (i) administering to said subject a therapeuticallyeffective amount of a composition comprising a naked or conjugatedanti-AFP MAb or fragment thereof or antibody fusion protein or fragmentthereof, of any one of the antibodies, fusion proteins, or fragmentsthereof of the present invention, (ii) formulating said anti-AFP MAb orfragment thereof or antibody fusion protein or fragment thereof in apharmaceutically suitable excipient, is contemplated. Preferably, such acomposition further comprises a second antibody, fusion prtoein, orfragment thereof. The second antibody, fusion protein, or fragmentthereof may or may not be an anti-AFP antibody, fusion protein orframent thereof. Also preferred, the anti-AFP antibody, fusion protein,or fragment thereof is an Immu31 antibody, fusion protein, or fragmentthereof. The preferred mode of administration is parenterally. Alsopreferred, the dosage is repeatedly administered. Still preferred, theanti-AFP antibody is administered in a dosage of 20 to 2000 milligramsprotein per dose.

[0116] The compositions for treatment contain at least one naked murine,humanized, chimeric or human anti-AFP antibody or fragment thereof aloneor in combination with other anti-AFP antibodies or antibody fragmentsthereof, such as other anti-AFP humanized, chimeric or human antibodies.Preferably, the anti-AFP antibody, fusion protein, or fragment thereofin the composition for treatment is administered in a dosage of 20-2000miligrams per dose. Also preferred, the anti-AFP antibody or fragmentthereof in the composition for treatment is an Immu31 antibody orfragment thereof. The present invention also contemplates treatment withat least one naked humanized, chimeric or human anti-AFP antibody orfragment thereof in combination with other antibodies or antibodyfragments thereof that are not anti-AFP antibodies, whereby these otherantibodies can be administered unconjugated (naked) or conjugated withat least one diagnostic/detection or therapeutic agent. For example,other antibodies suitable for combination therapy include, but are notlimited to, carcinoma-associated antibodies and fragments thereof suchas antibodies CEA, EGP-1, EGP-2 (e.g., 17-1A), MUC-1, MUC-2, MUC-3,MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, Ep-CAM, Tn,and Thomson-Friedenreich antigens, tumor necrosis antigens, tenascin, anoncogene, an oncogene product, IL-6, IGF-1, IGFR-1, tumor angiogenesisantigens, such as vascular endothelium growth factor (VEGF), placentalgrowth factor (PlGF), ED-B fibronectin, and against other vasculargrowth factors, Ga 733, ferritin and acidic isoferritin (AIF) of primaryhepatic carcinoma, or a combination thereof. Suitable antibodies couldalso include those targeted against oncogene markers or products, orantibodies against tumor-vasculature markers, such as the angiogenesisfactor, VEGF, and antibodies against certain immune response modulators,such as antibodies to CD40. Additionally, treatment can be effected withat least one humanized, chimeric or human anti-AFP immunoconjugate orfragment thereof alone or in combination with another anti-AFPantibodies or antibody fragments thereof, such as other anti-AFPhumanized, chimeric or human antibody. Preferably, the anti-AFP antibodyis a fragment thereof is an Immu31 antibody or fragment thereof. Stillpreferred, compositions for treatment can contain at least onehumanized, chimeric or human anti-AFP immunoconjugate or fragmentthereof in combination with other antibodies or antibody fragmentsthereof that are not anti-AFP antibodies, these being either naked orconjugated to a therapeutic agent. Such non-anti-AFP antibodies

[0117] Similarly, conjugated and naked anti-AFP humanized, chimeric orhuman antibodies or fragments thereof may be used alone or may beadministered with, but unconjugated to, the various diagnostic/detectionor therapeutic agents described herein. Also, naked or conjugatedanti-AFP antibodies to the same or different epitope or antigen may bealso combined with one or more of the antibodies of the presentinvention. Preferably, the anti-AFP antibody or fragment thereof is anImmu31 antibody or fragment thereof.

[0118] Accordingly, the present invention contemplates theadministration of murine, humanized, chimeric and human Immu31antibodies and fragments thereof alone, as a naked antibody, oradministered as a multimodal therapy. Multimodal therapies of thepresent invention further include immunotherapy with naked or conjugatedanti-AFP antibodies supplemented with administration of other conjugatedor unconjugated antibnody, fusion protein, or fragment thereof. Forexample, a humanized, chimeric or human Immu31 antibody may be combinedwith another naked humanized, naked chimeric or naked human Immu31antibody, or a humanized, chimeric or human Immu31 antibodyimmunoconjugate, such as a humanized, chimeric or human Immu31 antibodyconjugated to an isotope, one or more chemotherapeutic agents,cytokines, enzymes, enzyme-inhibitors, hormones or hormone antagonists,metals, toxins, or a combination thereof. A fusion protein of a murine,humanized, chimeric or human Immu31 antibody and a toxin or may also beused in this invention. Many different antibody combinations may beconstructed, either as naked antibodies or as partly naked and partlyconjugated with a therapeutic agent or immunomodulator, or merely incombination with another therapeutic agents, such as a cytotoxic drug orwith radiation.

[0119] The compositions for treatment contain at least one murine,humanized, chimeric or human monoclonal anti-AFP antibody or fragmentthereof alone or in combination with other antibodies and fragmentsthereof, such as other naked or conjugated, murine, humanized, chimeric,or human antibodies, or fragments thereof, or fusion proteins orfragments thereof, or therapeutic agents. In particular, combinationtherapy with a fully human antibody is also contemplated and is producedby the methods as set forth above.

[0120] Naked or conjugated antibodies, fusion proteins, or fragmentsthereof may be also combined with one or more of the antibodies, fusionproteins, or fragments thereof to the same or different epitope orantigen. For example, a naked, murine, humanized, chimeric or humanImmu31 antibody may be combined with a naked murine, humanized, nakedchimeric or naked human Immu31 antibody; a murine, humanized, chimericor human naked Immu31 antibody may be combined with a Immu31immunoconjugate; a naked murine, humanized, chimeric, human Immu31antibody may be combined with a different antibody radioconjugate or adifferent naked antibody; a murine, humanized, chimeric or fully humanImmu31 antibody may be combined with a murine, humanized, chimeric orhuman Immu31 antibody conjugated to an isotope, or to one or morechemotherapeutic agents, cytokines, toxins, enzymes, enzyme inhibitors,hormones, hormone antagonists, or a combination thereof. A fusionprotein of a murine, humanized, chimeric or human Immu31 antibody and atoxin or immunomodulator may also be used in this invention. Manydifferent antibody combinations, targeting at least two differentantigens may be constructed, either as naked antibodies or as partlynaked and partly conjugated with a therapeutic agent or immunomodulator,or merely in combination with another therapeutic agents, such as acytotoxic drug or with radiation.

[0121] Multimodal therapies of the present invention further includeimmunotherapy with naked Immu31 antibodies or fragments thereofsupplemented with administration of carcinoma associated antibodies inthe form of a conjugated or unconjugated antibody, fusion proteins, orfragment thereof. In a preferred embodiment, antibodies or fragmentsthereof for multimodal therapy include, but are not limited to,antibodies against CEA, EGP-1, EGP-2 (e.g., 17-lA), MUC-1, MUC-2, MUC-3,MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, Ep-CAM, Tn,and Thomson-Friedenreich antigens, tumor necrosis antigens, tenascin, anoncogene, an oncogene product, IL-6, IGF-1, IGFR-1, tumor angiogenesisantigens, such as vascular endothelium growth factor (VEGF), placentalgrowth factor (P1GF), ED-B fibronectin, and other vascular growthfactors, Ga 733, ferritin and acidic isoferritin (AIF) of primaryhepatic carcinoma, or a combination thereof. These antibodies includepolyclonal, monoclonal, chimeric, human or humanized antibodies andfragments thereof that recognize at least one epitope on these antigenicdeterminants.

[0122] In another form of multimodal therapy, subjects receive nakedanti-AFP antibodies or fragments thereof, andlor anti-AFPimmunoconjugates or fragments thereof, in conjunction with standardcancer chemotherapy. Preferably, the anti-AFP antibody or fragmentthereof is an Immu31 antibody or fragment thereof. 5-fluorouracil incombination with folinic acid, alone or in combination with irinotecan(CPT-11), is a regimen used to treat colorectal cancer. Other suitablecombination chemotherapeutic regimens are well known, such as withoxaliplatin alone, or in combination with these other drugs, to those ofskill in the art. In ovarian cancer, still other chemotherapeutic agentsmay be preferred, such as any one of the taxanes and platinum agents,Thio-TEPA and other alkylating agents (e.g., chlorambucil), as well asgemcitabine and other more recent classes of cytotoxic drugs. In apreferred multimodal therapy, both chemotherapeutic drugs and cytokinesare co-administered with a conjugated or unconjugated anti-AFP antibody,fusion protein, or fragment thereof, according to the present invention.Preferably, the anti-AFP antibody or fragment thereof is an Immu31antibody or fragment thereof. The cytokines, chemotherapeutic drugs andantibody, fusion protein, or fragment thereof, can be administered inany order, or together.

[0123] The present invention also encompasses the use of the bsAb and atleast one therapeutic or diagnostic/detection agent associated with thelinker moieties discussed above in intraoperative, intravascular, andendoscopic tumor and lesion detection, biopsy and therapy as describedin U.S. Pat. No. 6,096,289, and incorporated herein by reference.Preferably, the bispecific antibody has at least one arm that binds theAFP antigen, and more preferably, the Immu31 epitope.

[0124] The anti-AFP antibodies, fusion proteins, and fragments thereofof the present invention can be employed not only for therapeutic orimaging purposes, but also as aids in performing research in vitro. Forexample, the bsAbs of the present invention can be used in vitro toascertain if a targetable construct can form a stable complex with oneor more bsAbs. Such an assay would aid the skilled artisan inidentifying targetable constructs which form stable complexes withbsAbs. This would, in turn, allow the skilled artisan to identifytargetable constructs which are likely to be superior as therapeuticand/or imaging agents. Preferably, the anti-AFP antibody, fusionprotein, or fragment thereof is an Immu31 antibody, fusion protein, orfragment thereof.

[0125] The assay is advantageously performed by combining the targetableconstruct in question with at least two molar equivalents of a bsAb.Following incubation, the mixture is analyzed by size-exclusion HPLC todetermine whether or not the construct has bound to the bsAb.Alternatively, the assay is performed using standard combinatorialmethods wherein solutions of various bsAbs are deposited in a standard96-well plate. To each well, is added solutions of targetableconstruct(s). Following incubation and analysis, one can readilydetermine which construct(s) bind(s) best to which bsAb(s).

[0126] It should be understood that the order of addition of the bsAb tothe targetable construct is not crucial; that is, the bsAb may be addedto the construct and vice versa. Likewise, neither the bsAb nor theconstruct needs to be in solution; that is, they may be added either insolution or neat, whichever is most convenient. Lastly, the method ofanalysis for binding is not crucial as long as binding is established.Thus, one may analyze for binding using standard analytical methodsincluding, but not limited to, FABMS, high-field NMR or otherappropriate method in conjunction with, or in place of, size-exclusionHPLC.

[0127] Bispecific Antibody Therapy and Diagnosis

[0128] The present invention provides a bispecific antibody or antibodyfragment having at least one binding region that specifically binds atargeted cell marker and at least one other binding region thatspecifically binds a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment.

[0129] For example, a method of treating or identifying diseased tissuesin a subject, comprising: (A) administering to said subject abi-specific antibody or antibody fragment having at least one arm thatspecifically binds a targeted tissue and at least one other arm thatspecifically binds a targetable conjugate, wherein said one arm thatspecificially binds a targeted tissue is an Immu31 antibody; (B)optionally, administering to said subject a clearing composition, andallowing said composition to clear non-localized antibodies or antibodyfragments from circulation; (C) administering to said subject a firsttargetable conjugate which comprises a carrier portion which comprisesor bears at least one epitope recognizable by said at least one otherarm of said bi-specific antibody or antibody fragment, and one or moreconjugated therapeutic or diagnostic agents; and (D) when saidtherapeutic agent is an enzyme, further administering to said subject 1)a prodrug, when said enzyme is capable of converting said prodrug to adrug at the target site; or 2) a drug which is capable of beingdetoxified in said subject to form an intermediate of lower toxicity,when said enzyme is capable of reconverting said detoxified intermediateto a toxic form, and, therefore, of increasing the toxicity of said drugat the target site, or 3) a prodrug which is activated in said subjectthrough natural processes and is subject to detoxification by conversionto an intermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 4) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by said atleast one other arm of said bi-specific antibody or antibody fragment,and a prodrug, when said enzyme is capable of converting said prodrug toa drug at the target site, is described. Optionally, when said firsttargetable conjugate comprises a prodrug, administering a secondtargetable conjugate which comprises a carrier portion which comprisesor bears at least one epitope recognizable by said at least one otherarm of said bi-specific antibody or antibody or antibody fragment, andan enzyme capable of converting said prodrug to a drug or ofreconverting a detoxified intermediate of said drug to a toxic form.Preferably, the targetable conjugate comprises at least two HSG haptens.

[0130] In a related vein, a method for detecting or treating tumorsexpressing AFP in a mammal is described. This method comprises (A)administering an effective amount of a bispecific antibody or antibodyfragment comprising at least one arm that specifically binds a targetedtissue and at least one other arm that specifically binds a targetableconjugate, wherein said one arm that specifically binds a targetedtissue is an Immu31 antibody or fragment thereof, and (B) administeringa targetable conjugate selected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)—NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)—NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)—NH₂;

[0131] Optionally, the method further comprises administering to asubject a clearing composition, and allowing the composition to clearnon-localized antibodies or antibody fragments from the circulation.

[0132] Bispecific antibodies and fragments thereof of the presentinvention are useful in pretargeting methods and provide a preferred wayto deliver two therapeutic agents or two diagnostic/detection agents toa subject. U.S. Ser. No. 09/382,186 discloses a method of pretargetingusing a bispecific antibody, in which the bispecific antibody is labeledwith ¹²⁵I and delivered to a subject, followed by a divalent peptidelabeled with ^(99m)Tc. The delivery results in excellent tumor/normaltissue ratios for ¹²⁵I and ^(99m)Tc, thus showing the utility of twodiagnostic radioisotopes. Any combination of known therapeutic agents ordiagnostic agents can be used to label the Immu31 antibodies, Immu31fusion proteins, and fragments thereof of the present invention. Thebinding specificity of the Immu31 immunoconjugate, the efficacy of thetherapeutic agent or diagnostic agent and the effector activity of theFc portion of the antibody can be determined by standard testing of theconjugates.

[0133] The administration of a bsAb and a therapeutic agent associatedwith the linker moieties discussed above may be conducted byadministering the bsAb at some time prior to administration of thetherapeutic agent which is associated with the linker moiety. The dosesand timing of the reagents can be readily devised by a skilled artisan,and are dependent on the specific nature of the reagents employed. If absAb-F(ab′)₂ derivative is given first, then a waiting time of 24-72 hrbefore administration of the linker moiety would be appropriate. If anIgG-Fab′ bsAb conjugate is the primary targeting vector, then a longerwaiting period before administration of the linker moiety would beindicated, in the range of 3-10 days.

[0134] After sufficient time has passed for the bsAb to target to thediseased tissue, the diagnostic/detection agent is administered.Subsequent to administration of the diagnostic/detection agent, imagingcan be performed. Tumors can be detected in body cavities by means ofdirectly or indirectly viewing various structures to which energy of theappropriate wavelength is delivered and then collected. Lesions at anybody site can be viewed so long as nonionizing radiation or energy canbe delivered and recaptured from these structures. For example, PETwhich is a high resolution, non-invasive, imaging technique can be usedwith the inventive antibodies for the visualization of human disease. InPET, 511 keV gamma photons produced during positron annihilation decayare detected.

[0135] The linker moiety may also be conjugated to an enzyme capable ofactivating a prodrug at the target site or improving the efficacy of anormal therapeutic by controlling the body's detoxification pathways.Following administration of the bsAb, an enzyme conjugated to the linkermoiety, a low MW hapten recognized by the second arm of the bsAb, isadministered. After the enzyme is pretargeted to the target site, acytotoxic drug is injected, which is known to act at the target site.The drug may be one which is detoxified by the mammal's ordinarydetoxification processes. For example, the drug may be converted intothe potentially less toxic glucuronide in the liver. The detoxifiedintermediate can then be reconverted to its more toxic form by thepretargeted enzyme at the target site. Alternatively, an administeredprodrug can be converted to an active drug by the pretargeted enzyme.The pretargeted enzyme improves the efficacy of the treatment byrecycling the detoxified drug. This approach can be adopted for use withany enzyme-drug pair.

[0136] The enzyme capable of activating a prodrug at the target site orimproving the efficacy of a normal therapeutic by controlling the body'sdetoxification pathways may aternatively be conjugated to the hapten.The enzyme-hapten conjugate is administered to the subject followingadministration of the pre-targeting bsAb and is directed to the targetsite. After the enzyme is localized at the target site, a cytotoxic drugis injected, which is known to act at the target site, or a prodrug formthereof which is converted to the drug in situ by the pretargetedenzyme. As discussed above, the drug is one which is detoxified to forman intermediate of lower toxicity, most commonly a glucuronide, usingthe mammal's ordinary detoxification processes. The detoxifiedintermediate, e.g., the glucuronide, is reconverted to its more toxicform by the pretargeted enzyme and thus has enhanced cytotoxicity at thetarget site. This results in a recycling of the drug. Similarly, anadministered prodrug can be converted to an active drug through normalbiological processess. The pretargeted enzyme improves the efficacy ofthe treatment by recycling the detoxified drug. This approach can beadopted for use with any enzyme-drug pair.

[0137] The invention further contemplates the use of the inventive bsAband the diagnostic agent(s) in the context of Boron Neutron CaptureTherapy (BNCT) protocols. BNCT is a binary system designed to deliverionizing radiation to tumor cells by neutron irradiation oftumor-localized ¹⁰B atoms. BNCT is based on the nuclear reaction whichoccurs when a stable isotope, isotopically enriched ¹⁰B (present in19.8% natural abundance), is irradiated with thermal neutrons to producean alpha particle and a ⁷Li nucleus. These particles have a path lengthof about one cell diameter, resulting in high linear energy transfer.Just a few of the short-range 1.7 MeV alpha particles produced in thisnuclear reaction are sufficient to target the cell nucleus and destroyit. Success with BNCT of cancer requires methods for localizing a highconcentration of ¹⁰B at tumor sites, while leaving non-target organsessentially boron-free. Compositions and methods for treating tumors insubjects using pre-targeting bsAb for BNCT are described in co-pendingPatent Appl. Ser. No. 09/205,243, incorporated herein in its entiretyand can easily be modified for the purposes of the present invention.

[0138] A clearing agent may be used which is given between doses of thebsAb and the linker moiety. The present inventors have discovered that aclearing agent of novel mechanistic action may be used with theinvention, namely a glycosylated anti-idiotypic (anti-Id) Fab′ fragmenttargeted against the disease targeting arm(s) of the bsAb. For example,anti-CSAp (Mu-9 Ab) x anti-peptide bsAb is given and allowed to accretein disease targets to its maximum extent. To clear residual bsAb, ananti-idiotypic (anti-Id) Ab to Mu-9 is given, preferably as aglycosylated Fab′ fragment. The clearing agent binds to the bsAb in amonovalent manner, while its appended glycosyl residues direct theentire complex to the liver, where rapid metabolism takes place. Thenthe therapeutic which is associated with the linker moiety is given tothe subject. The anti-Id Ab to the Mu-9 arm of the bsAb has a highaffinity and the clearance mechanism differs from other disclosedmechanisms (see Goodwin et al., ibid), as it does not involvecross-linking, because the anti-Id-Fab′ is a monovalent moiety.

[0139] Also contemplated herein is a kit useful for treating oridentifying diseased tissues in a subject comprising: (A) a bi-specificantibody or antibody fragment having at least one arm that specificallybinds a targeted tissue and at least one other arm that specificallybinds a targetable conjugate, wherein said one arm that specificallybinds a targeted tissue is an Immu31 antibody or fragment thereof; (B) afirst targetable conjugate which comprises a carrier portion whichcomprises or bears at least one epitope recognizable by said at leastone other arm of said bi-specific antibody or antibody fragment, and oneor more conjugated therapeutic or diagnostic agents; and (C) optionally,a clearing composition useful for clearing non-localized antibodies andantibody fragments; and (D) optionally, when said therapeutic agentconjugated to said first targetable conjugate is an enzyme, 1) aprodrug, when said enzyme is capable of converting said prodrug to adrug at the target site; or 2) a drug which is capable of beingdetoxified in said subject to form an intermediate of lower toxicity,when said enzyme is capable of reconverting said detoxified intermediateto a toxic form, and, therefore, of increasing the toxicity of said drugat the target site, or 3) a prodrug which is activated in said subjectthrough natural processes and is subject to detoxification by conversionto an intermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 4) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by said atleast one other arm of said bi-specific antibody or antibody fragment,and a prodrug, when said enzyme is capable of converting said prodrug toa drug at the target site. Preferably, the targetable conjugate isselected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)—NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)—NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)—NH₂;

[0140] A method of screeing for a targetable conjugate is alsodescribed, comprising (A) contacting said targetable construct with abi-specific antibody or antibody fragment having at least one arm thatspecifically binds a targeted tissue and at least one other arm thatspecifically binds said targetable conjugate to give a mixture, whereinsaid one arm that specifically binds a targeted tissue is a Immu31antibody or fragment thereof; and (B) optionally incubating saidmixture; and (C) analyzing said mixture.

[0141] The present invention further provides a method for imagingmalignant tissue or cells in a mammal expressing AFP; a method ofintraoperatively identifying/disclosing diseased tissues expressing AFP,in a subject; a method for endoscopic identification of diseased tissuesexpressing AFP, in a subject and a method for the intravascularidentification of diseased tissues expressing AFP, in a subject. Suchmethods comprise (A) administering an effective amount of a bispecificantibody or antibody fragment comprising at least one arm thatspecifically binds a targeted tissue expressing AFP and at least oneother arm that specifically binds a targetable conjugate, wherein saidone arm that specifically binds a targeted tissue is an Immu31 antibodyor fragment thereof; and (B) administering a targetable conjugateselected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)—NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)—NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)—NH₂;

[0142] Also considered herein is a method of detection of lesions duringan endoscopic, laparoscopic, intravascular catheter, or surgicalprocedure, wherein the method comprises: (A) injecting a subject who isto undergo such a procedure with a bispecific antibody F(ab)₂ or F(ab′)₂fragment, wherein the bispecific antibody or fragment has a firstantibody binding site which specifically binds to a AFP antigen, and hasa second antibody binding site which specifically binds to a hapten, andpermitting the antibody fragment to accrete at target sites; (B)optionally clearing non-targeted antibody fragments using agalactosylated anti-idiotype clearing agent if the bispecific fragmentis not largely cleared from circulation within about 24 hours ofinjection, and injecting a bivalent labeled hapten, which quicklylocalizes at the target site and clears through the kidneys; (C)detecting the presence of the hapten by close-range detection ofelevated levels of accreted label at the target sites with detectionmeans, within 48 hours of the first injection, and conducting saidprocedure, wherein said detection is performed without the use of acontrast agent or subtraction agent. Preferably, the hapten is labeledwith a diagnostic/detection radioisotope, a MRI image-enhancing agent ora fluorescent label.

[0143] In a related vein, a method for close-range lesion detection,during an operative, intravascular, laparoscopic, or endoscopicprocedure, wherein the method comprises: (A) injecting a subject to sucha procedure parenterally with an effective amount of an Immu31immunoconjugate or fragment thereof, (B) conducting the procedure within48 hours of the injection; (C) scanning the accessed interior of thesubject at close range with a detection means for detecting the presenceof said labeled antibody or fragment thereof; and (D) locating the sitesof accretion of said labeled antibody or fragment thereof by detectingelevated levels of said labeled antibody or fragment thereof at suchsites with the detection means, is also described. Q9. PharmaceuticallySuitable Excipients

[0144] The murine, humanized, chimeric and human Immu31 MAbs to bedelivered to a subject can consist of the MAb alone, immunoconjugate,fusion protein, or can comprise one or more pharmaceutically suitableexcipients, one or more additional ingredients, or some combination ofthese.

[0145] The conjugated or unconjugated anti-AFP antibodies and fragmentsthereof, or fusion proteins and fragments thereof, of the presentinvention can be formulated according to known methods to preparepharmaceutically useful compositions. Preferably, the anti-AFP antibodyor fragment thereof is an Immu31 antibody or fragment thereof. Sterilephosphate-buffered saline is one example of a pharmaceutically suitableexcipient. Other suitable excipients are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

[0146] The conjugated or unconjugated anti-AFP antibody, fusion protein,or fragments thereof of the present invention can be formulated forintravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the anti-AFP antibody or fragments isan Immu31 antibody or fragment thereof. Formulations for injection canbe presented in unit dosage form, e.g., in ampules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0147] Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic or diagnostic/detectionimmunoconjugate or naked antibody, fusion protein, or fragments thereof.Control release preparations can be prepared through the use of polymersto complex or adsorb the immunoconjugate or naked antibody. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992).The rate of release of an immunoconjugate or antibody from such a matrixdepends upon the molecular weight of the immunoconjugate or antibody,the amount of immunoconjugate, antibody within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

[0148] The conjugated or unconjugated anti-AFP antibody, fusion protein,or fragments thereof may also be administered to a mammal subcutaneouslyor even by other parenteral routes. Moreover, the administration may beby continuous infusion or by single or multiple boluses. In general, thedosage of an administered immunoconjugate, or naked antibody, fusionprotein or fragments thereof for humans will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Typically, it is desirable toprovide the recipient with a dosage of immunoconjugate, naked antibodyfusion protein, naked antibody, or fragments thereof that is in therange of from about 1 mg/kg to 20 mg/kg as a single intravenousinfusion, although a lower or higher dosage also may be administered ascircumstances dictate. This dosage may be repeated as needed, forexample, once per week for 4-10 weeks, preferably once per week for 8weeks, and more preferably, once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months. Thedosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

[0149] For purposes of therapy, the conjugated or unconjugated antibody,fusion protein, or fragment thereof is administered to a mammal in atherapeutically effective amount. Preferably, the anti-AFP antibody orfragment thereof is an Immu31 antibody or fragment thereof. A suitablesubject for the present invention is usually a human, although anon-human animal subject is also contemplated. An antibody preparationis said to be administered in a “therapeutically effective amount” ifthe amount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient mammal. In particular, anantibody preparation of the present invention is physiologicallysignificant if its presence invokes an antitumor response or mitigatesthe signs and symptoms of an autoimmune disease state. A physiologicallysignificant effect could also be the evocation of a humoral and/orcellular immune response in the recipient mammal.

[0150] 10. Expression Vectors

[0151] The DNA sequence encoding a murine, humanized, chimeric or humanImmu31 MAb can be recombinantly engineered into a variety of known hostvectors that provide for replication of the nucleic acid. These vectorscan be designed, using known methods, to contain the elements necessaryfor directing transcription, translation, or both, of the nucleic acidin a cell to which it is delivered. Known methodology can be used togenerate expression constructs the have a protein-coding sequenceoperably linked with appropriate transcriptional/translational controlsignals. These methods include in vitro recombinant DNA techniques andsynthetic techniques. For example, see Sambrook et al., 1989, MOLECULARCLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (New York);Ausubel et al., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons (New York). Also provided for in this invention is the deliveryof a polynucleotide not associated with a vector.

[0152] Vectors suitable for use in the instant invention can be viral ornon-viral. Particular examples of viral vectors include adenovirus, AAV,herpes simplex virus, lentivirus, and retrovirus vectors. An example ofa non-viral vector is a plasmid. In a preferred embodiment, the vectoris a plasmid.

[0153] An expression vector, as described herein, is a polynucleotidecomprising a gene that is expressed in a host cell. Typically, geneexpression is placed under the control of certain regulatory elements,including constitutive or inducible promoters, tissue-specificregulatory elements, and enhancers. Such a gene is said to be “operablylinked to” the regulatory elements.

[0154] Preferably, the expression vector of the instant inventioncomprises the DNA sequence encoding a humanized, chimeric or humanImmu31 MAb, which includes both the heavy and the light chain variableand constant regions. However, two expression vectors may be used, withone comprising the heavy chain variable and constant regions and theother comprising the light chain variable and constant regions. Stillpreferred, the expression vector further comprises a promoter, a DNAsequence encoding a secretion signal peptide, a genomic sequenceencoding a human Ig light or heavy chain constant region, an Ig enhancerelement and at least one DNA sequence encoding a selection marker.

[0155] The invention is further described by reference to the followingexamples, which are provided for illustration only. The invention is notlimited to the examples but rather includes all variations that areevident from the teachings provided herein.

EXAMPLES Example 1. Molecular Cloning and Sequence Elucidation forImmu31Heavy and Light Chain Variable Regions

[0156] The VH and Vκ genes of Immu31 was obtained by RT-PCR as describedby Orlandi et al. (PNAS 86:3833-3837 (1989) and Leung et al. (Hybridoma13:469-476 (1994).

[0157] The total RNA was prepared from Immu31 hybridoma cells and RT-PCRwas performed to isolate the V genes as described (Leung et al.Hybridoma 13:469-476 (1994)). Briefly, the first strand cDNA was reversetranscribed from total RNA using the SuperScript preamplification system(GIBCO/BRL) in a reaction volume of 60 μl containing 20 μg of the RNAsannealed with 150 ng of random hexamer primer, 20 mM Tris-HCl, pH 8.4,15mM KCI, 2.5 mM MgCI_(2,5) mM dNTP mix, 10 mM DTT, 0.1 mg/ml BSA, and 600units of SuperScript reverse tmascriptase. The elongation step wasinitially allowed to proceed at room temperature for 10 min followed byincubation at 42° C. for 50 min. The reaction was terminated by heatingthe reaction mixture at 90° C. for 5 min. PCR reactions using the firststrand cDNA as templates were then carried out to amplify mouse Ig VHand Vκ genes. The Vκ sequence of Immu31 was amplified by using theprimer pair VK1BACK (Orlandi et al. PNAS 86:3833-3837 (1989) CK3′-BH(Leung et al. (Leung et al., 1993)). The resulting PCR products were˜350 bp. While the VH sequence was amplified with VH1BACK (Orlandi etal. PNAS 86:3833-3837 (1989)) and CH1-C (5′-AGCTGGGAAGGTGTGCAC-3′),which anneals to the CHI region of murine γ chains, resulting in PCRproducts of ˜500 bp. Both Vk and VH PCR fragments were cloned intopCR2.1 AT-cloning vector and the DNA sequences were determined by DNAsequencing (Sanger et al. PNAS 74:5463-5467 (1974)).

[0158] Multiple clones (8 for each) were selected for sequencing toeliminate possible errors resulted from PCR reaction. Majority of clonescontained an identical murine Ig VH (6) or Vκ (7) sequence, which wasdesignated as Immu31VH and Immu31Vκ, respectively (FIG. 1). The aminoacid sequences encoded by the genes were deduced and are also shown inFIG. 1. No defective mutations were identified within the sequences andimportant residues such as cycteines for intradomain disulfide linkageswere located at appropriate positions. Comparison with other mouse Vκsequences revealed that Immu31Vκ is a member of the kappa light chainsubclass V while Immu31VH belongs to mouse Ig heavy chain subclass IIA(Kabat et al., 1991).

Example 2. Construction of the Expression Vector for a Chimeric Immu31

[0159] To evaluate the “authenticity” of the cloned V gene segments, theputative murine Vκ and VH were constructed into a chimeric Immu31(cImmu31) containing human IgG and kappa constant domains and expressedin Sp2/0 cells. To facilitate subcloning of Immu31Vκ (FIG. 1A) togenerate the expression vector, the DNA sequence was modified at 3′endto include a BglII restriction site, AGATCT, by PCR amplification withprimers VK1BACK and VK1FOR (Orlandi et al. PNAS 86:3833-3837 (1989)).The resulting PCR product was digested with PvuII and BglII andforce-cloned into a pBR327-based staging vector (digested with PvuII andBclI), VKpBR, which contained Ig promoter, signal peptide sequence forsecretion and convenient restriction sites to facilitate in-frameligation of the VκPCR product (Leung et al.(Leung et al., 1994)).Similarly, the nucleotide sequences at positions 336-342 of hImmu31VH(FIG. 1B) were converted to BstEII site, GGTCACC, by PCR with primersVH1BACK and VH1FOR (Orlandi et al., 1989). The VH PCR product was thendigested with PstI and BstEII and ligated into PstI and BstEII digestedVHpBS, a pBluescript-based staging vector containing an Ig promoter, asignal peptide sequence and convenient restriction sites forin-frame-ligation of a VH sequence. The final V sequences in the cImmu31were designated as cImmu31VH and Vκ, confirmed by DNA sequencing andshown in FIG. 2A and 2B, respectively.

[0160] The fragments containing the VH and Vκ sequences of cImmu31,together with the promoter and signal peptide sequences, were excisedfrom the respective staging vectors, cImmu31VHpBS and cImmu31VKpBR, bydouble restriction-digestion with HindIII and BaniHI. The ca. 850 bpV_(H) fragment was then subcloned into the HindIII/BamHI site of amammalian expression vector, pG1g, in which cIMmu31VH was linked to thegenomic sequence of the human γ1 constant gene (Leung et al.(Leung etal., 1994)). Similarly, the ca. 650 bp Vκ fragment was inserted into theHindIII/BamHI site of pKh, which carrying the genomic gene sequence of ahuman κ constant region, an Ig enhancer, a κ enhancer, and thehygromycin-resistant gene as a marker for selection of transfectants(Leung et al.(Leung et al., 1994)). The final expression vectors weredesignated as cImmu31pG1g and cImmu31pKh, respectively.

Example 3. Transfection and Expression of Chimeric and Humanized Immu31

[0161] Same procedures were employed to express cImmu31 or hImmu31 inSp2/0 cells by transfection as described by Leung et al. (Hybridoma13:469-476 (1994)). As an example, expression of cImmu31 is describedhere. Briefly, linearized cImmu31pKh and cImmu31pG1g were co-transfectedinto Sp2/0 cells by electroporation. The transfected cells were grown in96-well plate for 2 days and then selected by the addition of hygromycinat a final concentration of 500 units/ml. The colonies began to emerge10-14 days after electroporation. Supernatants from colonies survivingselection were screened for the presence of mouse-human chimeric IgG byELISA. Briefly, supernatant samples from surviving clones were added intriplicate to ELISA microtiter plates precoated with goat anti-human(GAH) IgG, F(ab′)₂ fragment-specifc antibody (Jackson ImmunoResearch,West Grove, Pa.). The plates were incubated for 1 h at room temperature.Unbound proteins were removed by washing three times with washing buffer(PBS with 0.05% polysorbate-20). Horseradish peroxidase (HRP)-conjugatedGAH IgG, Fc fragment-specific antibody (Jackson ImmunoResearch) was thenadded to the wells. Following incubation for 1 h, the plates were washedsix times with washing buffer. A substrate solution containing 4 mM ofo-phenylenediamine dihydrochloride (OPD) and 0.04% H₂O₂, was added tothe wells. The reaction was allowed to proceed in the dark for 30 minand stopped by the addition of H₂SO₄ solution into each well beforemeasuring absorbance at 490 nm in an automated ELISA reader. Thepositive cell clones were expanded and cImmu31 was purified from cellculture supernatant by affinity chromatograpgy on a Protein A column. Acompetition Ag-binding assay was carried out to compare theimmunoreactivity of chimeric and murine Immu31 (Example 4). As shown inFIG. 3, cImmu31 and murine Immu31 competed equally well for the bindingof biotinylated murine Immu31 to the AFP antigen. These datademonstrated that the immunoreactivity of cImmu31 is comparable to thatof murine Immu31, thus confirming the authenticity of the Vκ and VHsequences obtained (FIG. 1).

[0162] Similar procedures were also used with another expression vector,pdHL2, as described in Example 5. Approximately 30 μg of hImmu31 pdHL2was linerized by digestion with SalI and transfected into Sp2/0 cells byelectroporation. The transfected cells were plated into 96-well plateand were allowed to recover for 2 days. After two days, MTX at a finalconcentration of 0.025 μM was added to the medium to selecttransfectants. MTX-resistant clones emerged in 2 weeks and Supernatantsfrom colonies surviving selection were monitored for human IgG secretionby ELISA as described above. Positive cell clones were expanded andhImmu31 was purified from cell culture supernatant by affinitychromatograpgy on a Protein A column.

Example 4. The Ag-binding Activity Assays

[0163] The Ag-binding activities of cImmu31 and hImmu3 Iwere determinedwith ELISA in ELISA microplate wells coated with AFP (Scripps ResearchInstitute, La Jolla, Calif.). Briefly, constant amount of biotinylatedmurine Immu31 was mixed with varying concentrations (0.01-100 μg/ml) oftesting Abs (Immu31, cImmu31 or hImmu31), and added into AFP-coatedmicrowells, and incubated at room temperature for 1 h. After washing,HRP conjugated streptavidin was added and incubated for 1 h at roomtemperature. The amount of HRP-conjugated streptavidin bound to theAFP-bound biotinylated Immu31 was revealed by reading OD at 490 nm in anELISA reader after the addition of a substrate solution containing 4 mMOPD and 0.04% H₂O₂.

Example 5. Choice of Human Frameworks and Sequence Design for hImmu31

[0164] By comparing the murine Immu31 V region FR sequences to that ofhuman Abs in the Kabat database (Sequences of Proteins of ImmunologicalInterest (Bethesda, MD: U.S. Departmet of Health and Human Services,Public Health Service, National Institute of Health, 1991), the FRs ofhuman REI and EU VH were found to exhibit the highest degree of sequencehomology to that of Immu31Vκ and Immu31VH, respectively (FIG. 4). Oneexception is the FR4 of Immu31VH, which showed the highest sequencehomology with that of NEWM VH (FIG. 4A). Thus, the FR sequences of RE1Vκ (FIG. 4B), FR1-3 of EU VH and FR4 of NEWM VH (FIG. 4A) were selectedas the scaffold for grafting the respective CDRs of Immu31. A few aminoacid residues in murine FRs that flank the putative CDRs were maintainedin hImmu31 based on the consideration that these residues have moreimpact on Ag binding than other FR residues. These residues are 5Q, 27Y,28A, 30T, 46Y, 48I, 66K, 67A and 94R of VH, and 4L, 39K, 48M, 49H, 58I,69R, 100G and 107K of Vκ. Additionally, based on the results of previoushumanization of LL2 (Leung et al. Mol. Immunol. 32:1413-1427 (1995)),two charged residues, 39K in FR2 and 69R in FR3 of Immu31Vκ, that havethe potential of CDR contacts and might affect the immunoreactivity ofthe resultant Ab were retained in the design of the humanized FRsequences (FIG. 4B). In order to evaluate the impact of the chargedmurine residues 39K and 69R on the binding activity of the Ab, twoalternate versions of humanized Vκ, hImmu31VKT39, and hImmu31VKT69, weredesigned by substituting either residue 39K or 69R with thecorresponding human residue, threonine, respectively (FIG. 4C).

[0165]FIGS. 3A compares the VH sequence of human EU with murine andhumanized Inmu31VH, and 3B compares human REI with murine and humanizedImmu31 Vκ. The dots indicate the residues in Immu31 and hImmu31sequences that are identical to the corresponding residues in the humanVH and Vκ sequences. FIG. 3C shows the difference between hImmu31Vκ andtwo variants, hImmu31VκT69 and hImmu31VκT39. The DNA and amino acidsequences of hImmu31VH and Vκ are shown in FIG. 5A and 5B, respectively.

Example 6. Expression and Characterization of hImmu31

[0166] The strategy as described by Leung et al. (Leung et al., 1994)was used to construct the designed Vκ and VH genes for hImmu31 using acombination of long oligonucleotide systheses and PCR. Each variablechain was constructed in two parts, a 5′- and 3′-half, designated as “A”and “B” respectively. Each half was produced by PCR amplification of asingle strand synthetic oligonucleotide template with two short flankingprimers, using Taq polyrnerase. The amplified fragments were firstcloned into the pCR2.1 TA cloning vector from Invitrogen (Carlsbad,Calif.) and subjected to DNA sequencing. The templates and primer pairsare listed as follows: Template Primers hImmu31VHA VHBACK/VHa hImmu31VHBVHb/VHFOR hImmu31VKA VKBACK/VKa hImmu31VKB VKb/VKFOR hImmu31VH domain

[0167] For the construction of the hImmu31VH domain, two longoligonucleotides, hImmu31VHA (135-mer) and hImmu31VHB (151-mer) weresynthesized on an automated DNA synthesizer (Applied Biosystem). Thesequence of long oligo hImmu31VHA represents the minus strand of thehImmu31VH domain complementary to nt 28 to 162 and that of hImmu31VHBwas complement to nt 181-331 as listed below.

[0168] hImmu31VHA (135 bp) 5′-GTAAGGATGA ATATATCCAA TCCAATACAGACCCTGTCCA GGTGCCTGCC TGACCCAGTG TATAACATAG CTAGTAAAAG CGTAGCCAGAAGCCTTGCAG GAGACCTTCA CTGATGACCC AGGTTTCTTG ACTTC-3′

[0169] hImmu31VHB (151 bp) 5′-CTTGGCCCCA GTAAGCAAAA GGGTCTCCCCCCCCAGATCT TGCACAAAAA TAAAATGCCG TGTCCTCAGA CCTCAGGCTG CTCAGCTCCATGTAGGCTGT ATTGGTGGAT TCGTCAGCTG TTATTGTGGC CTTGCCTTTG AACTTCTCAT T-3′

[0170] hImmu31VHA was amplified by PCR with a pair of primers VHBACK andVHa, while hImmu31VHB was amplified with VHb and VHFOR. The sequences ofthese primers are listed below: VHBACK 5′-CAGCTGCAGC AATCAGGGGCTGAAGTCAAG AAACCTG-3′ VHa 5′-GTACTTGGTA CCACCATTGT AAGGATGAAT ATATCC-3′VHb 5′-AATGGTGGTA CCAAGTACAA TGAGAAGTTC AAAGGC-3′ VHFOR5-′GGAGACGGTG ACCAGGGAGC CTTGGCCCCA GTAAGC-3′

[0171] where underlined sequences represent the restriction sites, PstI,KpnI, KpnI and BstEII, respectively. The resulting double-stranded PCRproducts, VHA and VHB, were digested with PstI/KpnI and KpnI/BstEII,respectively, gel purified, and assembled into the PstI/BstEII sites ofthe heavy chain staging vector, VHpBS, forming the full length hImmu31VHgene (FIG. 5A). The humanized VH sequence was subcloned into the pG1gvector, and the resultant human IgG1 heavy chain expression vector wasdesignated as hImmu31pG1g.

[0172] hImmu31Vκ domain

[0173] Similarly, for the construction of hImmu31Vκ domain, longoligonucleotides hImmu31VKA and Immu31VKB were used as template toconstruct the Vκ gene. hImmu31VKA represents the minus strand of thehImmu31Vκ domain complementary to nt 23 to 135 and that of hImmu31VKBwas complementary to nt 155-306 of the designed hImmu31Vκ (FIG. 5B).

[0174] hImmu31VKA (113 bp) 5′-TTTAGGTGCT TTCCCTGGTT TCTGCTGGTACCAACCTATA TACTTGTTAA TGTCTTGGCT TGCCTTACAA GTGATAGTGA CCCTATCTCCAACAGATGCG CTCAGAGATG ATG-3′

[0175] hImmu31VKB (152 bp) 5′-CTTGGTCCCT CCACCGAACG TCCACAGATCATCATACTGT AGACAATAAT ATGTTGCAAT GTCTTCTGGT TGAAGAGAGC TGATGGTGAAAGTATAATCT GTCCCAGATC CGCTGCCAGA GAATCGCGAA GGGATACCTG GCAGTAATGC AG-3′

[0176] hImmu31VKA was PCR-amplified with the primer pair of VKBACK andVKa, while hImmu31VKB was amplified with VKb and VKFOR. The sequences ofthese primers are listed below: VKBACK 5′-GAC ATT CAG CTG ACC CAG TCTCCA TCA TCT CTG AGC GC-3′ VKa 5′-A TGT GTA ATG CAT CAG CAG TTT AGG TGCTTT CC-3′ VKb 5′-CTG CTG ATG CAT TAC ACA TCT GCA TTA CTG CCA GG-3′ VKFOR5′-GA CCG GCA GAT CTG CAG CTT GGT CCC TCC AC-3′

[0177] The underlined sequences in VKBACK, VKa, VKb, and VKFOR representPvuII, NsiI, NsiI and BglII restriction sites, respectively. Theresulting double-stranded PCR products, VKA and VKB, were digested withPvuII/NsiI and NsiI/BglII, respectively, gel purified, and assembledinto the PvuII/BclI sites of the light chain staging vector, VKpBR.Finally, the humanized Vκ sequence was subcloned into the light chainexpression vector, pKh, forming hImmu31pKh.

[0178] hImmu31VκT39 and hImmu31VκT69 were similarly constructed and thefinal expression vectors for these two variants were hImmu31T39pKh andhImmu31T69pKh, respectively.

[0179] The Final Expression Vector for hImmu31

[0180] Using the two-expression vector system described above, i.e. pG1gand pKh, is preferred in the initial stage of humanization because itprovides flexibility of testing various combinations of Vκ and VHconstructs. The defective designs, if any, residing in the individualheavy or light chain can be systematically identified and corrected bymixing and matching each of the humanized chains with their chimericpartners. However, the transfected cells generated from the pG1g/pKhsystem typically produce antibodies at a level of less than 1 mg/literof terminal culture. To generate high-level antibody-producing celllines, a single expression vector, pdHL2, is preferred for theproduction of hImmu31. pdHL2 contains the expression cassettes for bothhuman IgG heavy and light chains under the control of IgH enhancer andMT₁ promoter, as well as a mouse dhfr gene, controlled by a weak SV40promotor, as a marker for selection of transfectants andco-amplification of the trans-genes (Gillies et al., J. Immunol. Methods125:191 (1989); Losman et al., Cancer 80:2660 (1997)). By replacing theVκ and VH segments of pdHL2, different chimeric or humanized Abs can beexpressed.

[0181] To construct the pdHL2 expression vector for hImmu31, hImmu31VHand Vκgene segments were subcloned into another set of staging vectors,VHpBS2 and VKpBR2, respectively. VHpBS2 is a modified staging vector ofVHpBS (Leung et al., Hybridoma, 13:469 (1994)), into which a XhoIrestriction site was introduced at 16 bases upstream of the translationinitiation codon. Similarly, VKpBR2 is a modified staging vector ofVKpBR (Leung et al., Hybridoma, 13:469 (1994)), into which a XbaIrestriction site was introduced at 14 bases upstream of the translationinitiation codon. The final expression vector hImmu31pdHL2 wasconstructed by sequencially subcloning the XbaI-BamHI and XhoI/BamHIfragments of hImmu31Vk and VH, respectively, into pdHL2. The finalexpression vector was designated as hImu31pdHL2.

[0182] Expression and Binding Activity Assays for hImmu31

[0183] The methods for transfection, screening positive transfectedclones and binding activity assays for hImmu31 were same as describedfor cImmu31 (see Example 3).

[0184] Three versions of the humanized Ab, hImmu31, hImmu31T39 andhImmu31T69, were expressed in Sp2/0 cells by co-transfection of theheavy chain expression vector, hImmu31pG1g, with either of the kappachain expression vectors: hImmu31pKh, hImmu31T39pKh or hImmu31T69pKh.The Ag-binding activities of these humanized Abs were evaluated by thesame competitive binding assay. While the AFP binding affinity ofhImmu31 and hImmu31T69 was similar to that of murine Immu31 or cImmu31,judging from their comparable competition with biotin-Immu31 (FIG. 6A),hImmmu31T39 was somewhat inferior (FIG. 6B). These results demonstratedthe successful humanization of Immu31 and revealed that the murine kappachain FR residue K³⁹ but not R⁶⁹ is important for maintaining theimmunoreactivity of Immu31.

[0185] The typical productivity of Abs from transfected Sp2/0 cells byusing pKh and pG1g expression vector system is in the single digit rangeof milligram per liter, which is practically insufficient for productionof large quantities of Abs for clinical applications. In the case ofhImmu31, the highest productivity of the selected clone co-transfectedwith hImmu31pKh and hImmu31pG1g was 2-3 mg/L. To increase the capabilityof the transfected cells to produce hImmu31, the heavy and kappa chainexpression cassettes were re-constructed into one single expressionvector, pdHL2, which contains the murine dhfr gene and allows forsubsequent amplification of the transfected gene products with stepwiseincrease of MTX concentrations. Three hImmu31pdHL2 transfected clones,314.2C11, 322.1G4 and 323.2H2, that were initially selected with 25 nMMTX and estimated to be producing 4, 15 and 8 mg/L of hImmu31,respectively, were subjected to amplification using procedures asdescribed by Losman et al. (Cancer 80:2660 (1997)). As the MTXconcentration in the cell culture medium gradually increased from 0.1 to3 μM, the productivity of hImmu31 from these cells was increasedconcomitantly and finally exceeded 100 mg/L in termination roller bottlecultures (data not shown). The purified hImmu31 fromhImmu31pdHL2-transfected cells showed comparable immunoreactivity asthat of its murine and chimeric counterparts (FIG. 6C).

Example 7. Therapy of a Patient with Hepatocelluar Carcinoma withRadiolabeled Humanized Anti-AFP Monoclonal Antibody.

[0186] A 57-year-old man presenting with jaundice, malaise, loss ofweight, and general weakness, is diagnosed with an inoperablehepatocellular carcinoma that appears by computed tomography to extendabout 6 cm in diameter in the right lobe of the liver, and to alsoappear as a single 3-cm lesion in the left lobe. His serum AFP level atthe time of presentation measures 150 ng/mL, with a 40% increase in hisserum transaminase and bilirubin levels, and a 50% increase in his serumLDH level. The right lobe lesion is confirmed by biopsy to behepatocellular carcinoma expressing AFP. The patient is then given twocycles of humanized Immu31 monoclonal antibody conjugated by DOTA with90-Y, so that an infusion is administered for each therapy of a dose of25 mCi (100 mg antibody protein). The first therapy is given in anoutpatient setting, and is repeated 6 weeks later. Prior to eachtherapy, a diagnostic dose of 111-In conjugated by DOTA to the antibodyis also injected in order to demonstrate tumor targeting and to estimatethe radiation dose delivered to the tumor and to other normal tissues,such as liver, kidney and bone marrow, so that the therapeutic dose with90-Y, given a week later, can be adjusted so as not to induce normaltissue/organ toxicity beyond what is considered tolerable (e.g., 2000cGy to kidneys). The patient is then monitored for response by repeatedcomputer tomography scans every 4-8 weeks post therapy, as well as byserum AFP, bilirubin, transaminase, and LDH levels. Eight weeks afterthe second therapeutic administration of the 90-Y-labeled antibody, hisserum levels of bilirubin, transaminases, and LDH decreases to about 20%above normal, and his serum AFP titer is measured at 60 ng/mL, whichalso constitutes an improvement. CT measurements of his liver diseaseshows an almost complete disappearance of the left lobe lesion and a 40%reduction of the larger mass in the right lobe. The patient then becamea candidate for surgical resection of his right lobe, since it isconsidered that the remaining small lesion in the left lobe is notcancer, but scar tissue. This is further confirmed by a diagnostic studyperformed with 111-In-labeled Immu31 antibody, which shows uptake in theright lobe mass but not in the left lobe, thus indicating that noAFP-expressing disease is in the left lobe.

[0187] All of the publications and patent applications and patents citedin this specification are herein incorporated in their entirety byreference.

[0188] Although the foregoing refers to particular preferredembodiments, it will be understood that the present invention is not solimited. It will occur to those of ordinary skill in the art thatvarious modifications may be made to the disclosed embodiments and thatsuch modifications are intended to be within the scope of the presentinvention, which is defined by the following claims.

1 48 1 11 PRT Mus sp. 1 Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Gly 1 510 2 7 PRT Mus sp. 2 Tyr Thr Ser Ala Leu Leu Pro 1 5 3 8 PRT Mus sp. 3Leu Gln Tyr Asp Asp Leu Trp Thr 1 5 4 5 PRT Mus sp. 4 Ser Tyr Val IleHis 1 5 5 17 PRT Mus sp. 5 Tyr Ile His Pro Tyr Asn Gly Gly Thr Lys TyrAsn Glu Lys Phe Lys 1 5 10 15 Gly 6 9 PRT Mus sp. 6 Ser Gly Gly Gly AspPro Phe Ala Tyr 1 5 7 4 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 7 Phe Lys Tyr Lys 1 8 16 DNAArtificial Sequence Description of Artificial Sequence Primer 8acagtcactg agctgg 16 9 36 DNA Artificial Sequence Description ofArtificial Sequence Primer 9 gccggatcct gactggatgg tgggaagatg gataca 3610 24 DNA Artificial Sequence Description of Artificial Sequence Primer10 gacattcagc tgacccagtc tcca 24 11 33 DNA Artificial SequenceDescription of Artificial Sequence Primer 11 ctcactggat ggtgggaagatggatacagt tgg 33 12 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 12 aggtsacarc tgcagsagtc wgg 23 13 30 DNAArtificial Sequence Description of Artificial Sequence Probe 13agactgcagg agagctggga aggtgtgcac 30 14 30 DNA Artificial SequenceDescription of Artificial Sequence Probe 14 gaagcacacg actgaggcacctccagatgt 30 15 15 PRT Artificial Sequence Description of ArtificialSequence Linker peptide 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser GlyGly Gly Gly Ser 1 5 10 15 16 4 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 16 Lys Tyr Lys Lys 1 17 18 DNAArtificial Sequence Description of Artificial Sequence Primer 17agctgggaag gtgtgcac 18 18 135 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 18 gtaaggatga atatatccaatccaatacag accctgtcca ggtgcctgcc tgacccagtg 60 tataacatag ctagtaaaagcgtagccaga agccttgcag gagaccttca ctgatgaccc 120 aggtttcttg acttc 135 19151 DNA Artificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 19 cttggcccca gtaagcaaaa gggtctcccc ccccagatcttgcacaaaaa taaaatgccg 60 tgtcctcaga cctcaggctg ctcagctcca tgtaggctgtattggtggat tcgtcagctg 120 ttattgtggc cttgcctttg aacttctcat t 151 20 37DNA Artificial Sequence Description of Artificial Sequence Primer 20cagctgcagc aatcaggggc tgaagtcaag aaacctg 37 21 36 DNA ArtificialSequence Description of Artificial Sequence Primer 21 gtacttggtaccaccattgt aaggatgaat atatcc 36 22 36 DNA Artificial SequenceDescription of Artificial Sequence Primer 22 aatggtggta ccaagtacaatgagaagttc aaaggc 36 23 36 DNA Artificial Sequence Description ofArtificial Sequence Primer 23 ggagacggtg accagggagc cttggcccca gtaagc 3624 113 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 24 tttaggtgct ttccctggtt tctgctggta ccaacctatatacttgttaa tgtcttggct 60 tgccttacaa gtgatagtga ccctatctcc aacagatgcgctcagagatg atg 113 25 152 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 25 cttggtccct ccaccgaacgtccacagatc atcatactgt agacaataat atgttgcaat 60 gtcttctggt tgaagagagctgatggtgaa agtataatct gtcccagatc cgctgccaga 120 gaatcgcgaa gggatacctggcagtaatgc ag 152 26 38 DNA Artificial Sequence Description ofArtificial Sequence Primer 26 gacattcagc tgacccagtc tccatcatct ctgagcgc38 27 33 DNA Artificial Sequence Description of Artificial SequencePrimer 27 atgtgtaatg catcagcagt ttaggtgctt tcc 33 28 35 DNA ArtificialSequence Description of Artificial Sequence Primer 28 ctgctgatgcattacacatc tgcattactg ccagg 35 29 31 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 29 gaccggcaga tctgcagctt ggtccctcca c 3130 351 DNA Artificial Sequence CDS (1)..(351) Description of ArtificialSequence Synthetic Immu31Vh nucleotide sequence 30 gtg aag ctg cag gagtca gga cct gaa ctg gta aag cct ggg gct tca 48 Val Lys Leu Gln Glu SerGly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 gtg aag atg tcc tgcaag gct tct gga tac gct ttc act agc tat gtt 96 Val Lys Met Ser Cys LysAla Ser Gly Tyr Ala Phe Thr Ser Tyr Val 20 25 30 ata cac tgg gtg agg cagaag cct ggg cag ggc ctt tat tgg att gga 144 Ile His Trp Val Arg Gln LysPro Gly Gln Gly Leu Tyr Trp Ile Gly 35 40 45 tat att cat cct tac aat ggtggt acc aag tac aat gag aag ttc aaa 192 Tyr Ile His Pro Tyr Asn Gly GlyThr Lys Tyr Asn Glu Lys Phe Lys 50 55 60 ggc aag gcc aca ctg act tca gacaaa tcg tcc agc aca acc tac atg 240 Gly Lys Ala Thr Leu Thr Ser Asp LysSer Ser Ser Thr Thr Tyr Met 65 70 75 80 gag ctc agc agc ctg acc tct gaggac tct gcg gtc tat tac tgt gca 288 Glu Leu Ser Ser Leu Thr Ser Glu AspSer Ala Val Tyr Tyr Cys Ala 85 90 95 aga tct ggg ggg gga gac cct ttt gcttac tgg ggc caa ggg act ctg 336 Arg Ser Gly Gly Gly Asp Pro Phe Ala TyrTrp Gly Gln Gly Thr Leu 100 105 110 gtc act gtc tct gca 351 Val Thr ValSer Ala 115 31 117 PRT Artificial Sequence Description of ArtificialSequence Synthetic Immu31Vh protein sequence 31 Val Lys Leu Gln Glu SerGly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Met Ser CysLys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr Val 20 25 30 Ile His Trp Val ArgGln Lys Pro Gly Gln Gly Leu Tyr Trp Ile Gly 35 40 45 Tyr Ile His Pro TyrAsn Gly Gly Thr Lys Tyr Asn Glu Lys Phe Lys 50 55 60 Gly Lys Ala Thr LeuThr Ser Asp Lys Ser Ser Ser Thr Thr Tyr Met 65 70 75 80 Glu Leu Ser SerLeu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ser Gly GlyGly Asp Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr ValSer Ala 115 32 321 DNA Artificial Sequence CDS (1)..(321) Description ofArtificial Sequence Synthetic Immu31Vk nucleotide sequence 32 gac attcag ctg acc cag tct cca tcc tca ctg tct gca tct ctg gga 48 Asp Ile GlnLeu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 ggc aaagtc acc atc act tgc aag gca agc caa gac att aac aag tat 96 Gly Lys ValThr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 ata ggt tggtac caa cac aag cct gga aaa ggt cct agg cta ctc atg 144 Ile Gly Trp TyrGln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Met 35 40 45 cat tac aca tctgca tta ctg cca ggc atc cca tca agg ttc agt gga 192 His Tyr Thr Ser AlaLeu Leu Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 agt ggg tct ggg agagat tat tcc ttc agc atc agc aac ctg gag cct 240 Ser Gly Ser Gly Arg AspTyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 gaa gat att gca acttat tat tgt cta cag tat gat gat ctg tgg acg 288 Glu Asp Ile Ala Thr TyrTyr Cys Leu Gln Tyr Asp Asp Leu Trp Thr 85 90 95 ttc ggt gga ggc acc aagctg gaa atg aaa cgg 321 Phe Gly Gly Gly Thr Lys Leu Glu Met Lys Arg 100105 33 107 PRT Artificial Sequence Description of Artificial SequenceSynthetic Immu31Vk protein sequence 33 Asp Ile Gln Leu Thr Gln Ser ProSer Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr CysLys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Gly Trp Tyr Gln His LysPro Gly Lys Gly Pro Arg Leu Leu Met 35 40 45 His Tyr Thr Ser Ala Leu LeuPro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp TyrSer Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr TyrTyr Cys Leu Gln Tyr Asp Asp Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr LysLeu Glu Met Lys Arg 100 105 34 354 DNA Artificial Sequence CDS(1)..(354) Description of Artificial Sequence Synthetic cImmu31Vhnucleotide sequence 34 cag gtc cag ctg cag gag tca gga cct gaa ctg gtaaag cct ggg gct 48 Gln Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val LysPro Gly Ala 1 5 10 15 tca gtg aag atg tcc tgc aag gct tct gga tac gctttc act agc tat 96 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ala PheThr Ser Tyr 20 25 30 gtt ata cac tgg gtg agg cag aag cct ggg cag ggc ctttat tgg att 144 Val Ile His Trp Val Arg Gln Lys Pro Gly Gln Gly Leu TyrTrp Ile 35 40 45 gga tat att cat cct tac aat ggt ggt acc aag tac aat gagaag ttc 192 Gly Tyr Ile His Pro Tyr Asn Gly Gly Thr Lys Tyr Asn Glu LysPhe 50 55 60 aaa ggc aag gcc aca ctg act tca gac aaa tcg tcc agc aca acctac 240 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Thr Tyr65 70 75 80 atg gag ctc agc agc ctg acc tct gag gac tct gcg gtc tat tactgt 288 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys85 90 95 gca aga tct ggg ggg gga gac cct ttt gct tac tgg ggc caa ggg acc336 Ala Arg Ser Gly Gly Gly Asp Pro Phe Ala Tyr Trp Gly Gln Gly Thr 100105 110 acg gtc acc gtc tcc tca 354 Thr Val Thr Val Ser Ser 115 35 118PRT Artificial Sequence Description of Artificial Sequence SyntheticcImmu31Vh protein sequence 35 Gln Val Gln Leu Gln Glu Ser Gly Pro GluLeu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala SerGly Tyr Ala Phe Thr Ser Tyr 20 25 30 Val Ile His Trp Val Arg Gln Lys ProGly Gln Gly Leu Tyr Trp Ile 35 40 45 Gly Tyr Ile His Pro Tyr Asn Gly GlyThr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser AspLys Ser Ser Ser Thr Thr Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr SerGlu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Gly Gly Asp ProPhe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 11536 321 DNA Artificial Sequence CDS (1)..(321) Description of ArtificialSequence Synthetic cImmu31Vk nucleotide sequence 36 gac atc cag ctg acccag tct cca tcc tca ctg tct gca tct ctg gga 48 Asp Ile Gln Leu Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 ggc aaa gtc acc atcact tgc aag gca agc caa gac att aac aag tat 96 Gly Lys Val Thr Ile ThrCys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 ata ggt tgg tac caa cacaag cct gga aaa ggt cct agg cta ctc atg 144 Ile Gly Trp Tyr Gln His LysPro Gly Lys Gly Pro Arg Leu Leu Met 35 40 45 cat tac aca tct gca tta ctgcca ggc atc cca tca agg ttc agt gga 192 His Tyr Thr Ser Ala Leu Leu ProGly Ile Pro Ser Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aga gat tat tccttc agc atc agc aac ctg gag cct 240 Ser Gly Ser Gly Arg Asp Tyr Ser PheSer Ile Ser Asn Leu Glu Pro 65 70 75 80 gaa gat att gca act tat tat tgtcta cag tat gat gat ctg tgg acg 288 Glu Asp Ile Ala Thr Tyr Tyr Cys LeuGln Tyr Asp Asp Leu Trp Thr 85 90 95 ttc ggt gga ggg acc aag ctg gag atcaaa cga 321 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 37 107PRT Artificial Sequence Description of Artificial Sequence SyntheticcImmu31Vk protein sequence 37 Asp Ile Gln Leu Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Lys AlaSer Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Gly Trp Tyr Gln His Lys Pro GlyLys Gly Pro Arg Leu Leu Met 35 40 45 His Tyr Thr Ser Ala Leu Leu Pro GlyIle Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser PheSer Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr CysLeu Gln Tyr Asp Asp Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu GluIle Lys Arg 100 105 38 117 PRT Artificial Sequence Description ofArtificial Sequence EUVH protein sequence 38 Pro Val Gln Leu Val Gln SerGly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser CysLys Ala Ser Gly Gly Thr Phe Ser Arg Ser 20 25 30 Ala Ile Ile Trp Val ArgGln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Val Pro MetPhe Gly Pro Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr IleThr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser SerLeu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys 85 90 95 Ala Gly Gly Tyr GlyIle Tyr Ser Pro Glu Glu Tyr Asn Gly Gly Leu 100 105 110 Val Thr Val SerSer 115 39 354 DNA Artificial Sequence CDS (1)..(354) Description ofArtificial Sequence Synthetic hImmu31Vh nucleotide sequence 39 cag gtccag ctg cag caa tca ggg gct gaa gtc aag aaa cct ggg tca 48 Gln Val GlnLeu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 tca gtgaag gtc tcc tgc aag gct tct ggc tac gct ttt act agc tat 96 Ser Val LysVal Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr 20 25 30 gtt ata cactgg gtc agg cag gca cct gga cag ggt ctg tat tgg att 144 Val Ile His TrpVal Arg Gln Ala Pro Gly Gln Gly Leu Tyr Trp Ile 35 40 45 gga tat att catcct tac aat ggt ggt acc aag tac aat gag aag ttc 192 Gly Tyr Ile His ProTyr Asn Gly Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 aaa ggc aag gcc acaata aca gct gac gaa tcc acc aat aca gcc tac 240 Lys Gly Lys Ala Thr IleThr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 atg gag ctg agc agcctg agg tct gag gac acg gca ttt tat ttt tgt 288 Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Phe Tyr Phe Cys 85 90 95 gca aga tct ggg ggg ggagac cct ttt gct tac tgg ggc caa ggc tcc 336 Ala Arg Ser Gly Gly Gly AspPro Phe Ala Tyr Trp Gly Gln Gly Ser 100 105 110 ctg gtc acc gtc tcc tca354 Leu Val Thr Val Ser Ser 115 40 118 PRT Artificial SequenceDescription of Artificial Sequence Synthetic hImmu31Vh protein sequence40 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 510 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Ser Tyr 2025 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Tyr Trp Ile 3540 45 Gly Tyr Ile His Pro Tyr Asn Gly Gly Thr Lys Tyr Asn Glu Lys Phe 5055 60 Lys Gly Lys Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 6570 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys85 90 95 Ala Arg Ser Gly Gly Gly Asp Pro Phe Ala Tyr Trp Gly Gln Gly Ser100 105 110 Leu Val Thr Val Ser Ser 115 41 321 DNA Artificial SequenceCDS (1)..(321) Description of Artificial Sequence Synthetic hImmu31Vknucleotide sequence 41 gac atc cag ctg acc cag tct cca tca tct ctg agcgca tct gtt gga 48 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val Gly 1 5 10 15 gat agg gtc act atc act tgt aag gca agc caa gacatt aac aag tat 96 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp IleAsn Lys Tyr 20 25 30 ata ggt tgg tac cag cag aaa cca ggg aaa gca cct aaactg ctg atg 144 Ile Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys LeuLeu Met 35 40 45 cat tac aca tct gca tta ctg cca ggt atc cct tcg cga ttctct ggc 192 His Tyr Thr Ser Ala Leu Leu Pro Gly Ile Pro Ser Arg Phe SerGly 50 55 60 agc gga tct ggg aca gat tat act ttc acc atc agc tct ctt caacca 240 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80 gaa gac att gca aca tat tat tgt cta cag tat gat gat ctg tggacg 288 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Trp Thr85 90 95 ttc ggt gga ggg acc aag ctg cag atc aaa cga 321 Phe Gly Gly GlyThr Lys Leu Gln Ile Lys Arg 100 105 42 107 PRT Artificial SequenceDescription of Artificial Sequence Synthetic hImmu31Vk protein sequence42 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 510 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 2025 30 Ile Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Met 3540 45 His Tyr Thr Ser Ala Leu Leu Pro Gly Ile Pro Ser Arg Phe Ser Gly 5055 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 6570 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Trp Thr85 90 95 Phe Gly Gly Gly Thr Lys Leu Gln Ile Lys Arg 100 105 43 108 PRTArtificial Sequence Description of Artificial Sequence REI proteinsequence 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser ValGly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile IleLys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Lys LeuLeu Ile 35 40 45 Tyr Glu Ala Ser Asn Leu Gln Ala Gly Val Pro Ser Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser LeuGln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln SerLeu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr Arg 100105 44 11 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide NEWMVH 44 Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser1 5 10 45 107 PRT Artificial Sequence Description of Artificial SequencehImmu31VkT69 protein sequence 45 Asp Ile Gln Leu Thr Gln Ser Pro Ser SerLeu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys AlaSer Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Gly Trp Tyr Gln Gln Lys Pro GlyLys Ala Pro Lys Leu Leu Met 35 40 45 His Tyr Thr Ser Ala Leu Leu Pro GlyIle Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr PheThr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr CysLeu Gln Tyr Asp Asp Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu GlnIle Lys Arg 100 105 46 107 PRT Artificial Sequence Description ofArtificial Sequence hImmu31VkT39 protein sequence 46 Asp Ile Gln Leu ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val ThrIle Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Gly Trp TyrGln Gln Thr Pro Gly Lys Ala Pro Lys Leu Leu Met 35 40 45 His Tyr Thr SerAla Leu Leu Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser GlyArg Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp IleAla Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Trp Thr 85 90 95 Phe Gly GlyGly Thr Lys Leu Gln Ile Lys Arg 100 105 47 4 PRT Artificial SequenceDescription of Artificial Sequence Synthetic linker peptide 47 Gly GlyGly Ser 1 48 4 PRT Artificial Sequence Description of ArtificialSequence Synthetic peptide 48 Phe Lys Tyr Lys 1

What is claimed:
 1. A monoclonal (MAb) antibody or fragment thereof thatbinds an alpha-fetoprotein (AFP) antigen.
 2. The monoclonal antibody orfragment thereof of claim 1, wherein said antibody or fragment thereofis a lmmu3 1 antibody.
 3. The monoclonal antibody or fragment thereof ofclaim 1, wherein said antibody or fragment thereof is humanized.
 4. Themonoclonal antibody or fragment thereof of claim 1, wherein saidantibody or fragment thereof is a chimeric antibody or fragment thereof.5. The monoclonal antibody or fragment thereof of claim 1, wherein saidantibody or fragment thereof is a fully human antibody or fragmentthereof.
 6. The antibody or fragment thereof of claim 3, comprising thecomplementarity-determining regions (CDRs) of a murine anti-AFP MAb andthe framework (FR) regions of the light and heavy chain variable regionsof a human antibody and the light and heavy chain constant regions of ahuman antibody, wherein the CDRs of the light chain variable region ofthe humanized AFP MAb comprises CDR1 comprising an amino acid sequenceof KASQDINKYIG; CDR2 comprising an amino acid sequence of YTSALLPandCDR3 comprising an amino acid sequence of LQYDDLWT; and the CDRs of theheavy chain variable region of the humanized AFP MAb comprises CDR1comprising an amino acid sequence of SYVIH; CDR2 comprising an aminoacid sequence of YIHPYNGGTKYNEKFKG and CDR3 comprising an amino acidsequence of SGGGDPFAY.
 7. The antibody or fragment thereof of claim 3,wherein the FRs of the light and heavy chain variable regions of saidantibody or fragment thereof comprise at least one amino acidsubstituted from the corresponding FRs of the murine anti-AFP antibodyor fragment thereof.
 8. The antibody or fragment thereof of claim 7,wherein said amino acid from said murine MAb is at least one amino acidselected from the group consisting of amino acid residue 5, 27, 28, 30,46, 48, 66, 67 and 94 of the murine heavy chain variable region of FIG.4A.
 9. The antibody or fragment thereof of claim 7, wherein said murineamino acids are at least one amino acid selected from the groupconsisting of amino acid residue 4, 39, 48, 49, 58, 69, 100 and 107 ofthe murine light chain variable region FIG. 4B.
 10. The antibody orfragment thereof of claim 2, wherein said antibody or fragment thereofcomprises the Immu31 VK nucleotide sequence of figure lB.
 11. Theantibody or fragment thereof of claim 2, wherein said antibody orfragment thereof comprises the Immu31 VH nucleotide sequence of FIG. 1A.12. The antibody or fragment thereof of claim 3, wherein said antibodyor fragment thereof comprises a hImmu3l VK nucleotide sequence of FIG.5B.
 13. The antibody or fragment thereof of claim 3, wherein saidantibody or fragment thereof comprises a hhmmu31 VH nucleotide sequenceof FIG. 5A.
 14. A CDR-grafted humanized heavy chain comprising thecomplementarity determining regions (CDRs) of a murine lmmu31 MAb andthe framework region of the heavy chain variable region of a humanantibody and the heavy chain constant region of a human antibody,wherein the CDRs of the heavy chain variable region of the humanizedanti-AFP MAb comprises CDRI comprising an amino acid sequence of SYVIH;CDR2 comprising an amino acid sequence of YIHPYNGGTKYNEKFKG and CDR3comprising an amino acid sequence of SGGGDPFAY.
 15. A CDR-graftedhumanized light chain comprising the complementarity determining regions(CDRs) of a murine lmmu31 MAb and the framework region of the lightchain variable region of a human antibody and the light chain constantregion of a human antibody, wherein the CDRs of the light chain variableregion of the humanized anti-AFP MAb comprises CDR1 comprising an aminoacid sequence of KASQDINKYIG; CDR2 comprising an amino acid sequence ofYTSALLP and CDR3 comprising an amino acid sequence of LQYDDLWT.
 16. TheAFP antibodies or fragment thereof of claim 1, wherein said fragment isselected from the group consisting of Fv, F(ab')₂, Fab' and Fab.
 17. Adiagnostic/detection or therapeutic immunoconjugate comprising anantibody component that comprises an AFP MAb or fragment thereof ofclaim 1 or an antibody fusion protein or fragment thereof that comprisesthe antibody of claim 1, wherein said antibody component is bound to atleast one diagnostic/detection agent or at least one therapeutic agent.18. The diagnostic/detection immunoconjugate of claim 17, wherein saiddiagnostic/detection agent comprises at least one photoactivediagnostic/detection agent.
 19. The diagnostic/detection immunoconjugateof claim 18, wherein said photoactive diagnostic agent comprises achromagen or dye.
 20. The diagnostic/detection immunoconjugate of claim17, wherein said diagnostic/detection agent is a radionuclide with anenergy between 20 and 10,000 keV.
 21. The diagnostic/detectionimmunoconjugate of claim 20, wherein said radionuclide is a gamma-,beta- or a positron-emitting isotope.
 22. The diagnostic/detectionimmunoconjugate of claim 21, wherein said radionuclide is selected fromthe group consisting of ¹⁸F, ⁵¹Mn, ⁵²mMn, ⁵²Fe, ⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁸Ga,⁷²As, ⁷⁵Br, ⁷⁶Br, ⁸²mRb, ⁸³Sr, ⁸⁶y, ⁸⁹Zr, ⁹⁴mTc, IIn, 120i, 124i, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷CU, ⁶⁷Ga, ⁷⁵Se, ⁹⁷Ru, ⁹⁹mTc, IllIn, 1¹⁴mIn, 123i,125i, 131i, ¹⁶⁹, 1⁹⁷Hg, and 21'Tl.
 23. The diagnostic/detectionimmunoconjugate of claim 17, wherein said diagnostic agent is a contrastagent.
 24. The diagnostic/detection immunoconjugate of claim 23, whereinsaid contrast agent is a paramagnetic ion.
 25. The diagnostic/detectionimmunoconjugate of claim 23, wherein said contrast agent is anultrasound-enhancing agent.
 26. The diagnostic/detection immunoconjugateof claim 25, wherein said ultrasound enhancing agent is a liposome thatis conjugated to a humanized Immu31 or fragment thereof.
 27. Thediagnostic/detection immunoconjugate of claim 26, wherein said liposomeis gas filled.
 28. A diagnostic/detection immunoconjugate of claim 24,wherein said paramagnetic ion comprises a metal selected from the groupconsisting of chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III).
 29. Thediagnostic/detection immunoconjugate of claim 23, wherein said contrastagent is a radiopaque compound.
 30. The diagnostic/detectionimmunoconjugate of claim 29, wherein said radiopaque compound isselected from the group consisting of iodine compounds, bariumcompounds, gallium compounds and thallium compounds.
 31. Thediagnostic/detection immunoconjugate of claim 17, wherein saidimmunoconjugate is used in intraoperative, endoscopic, or intravasculartumor detection/diagnosis.
 32. The therapeutic immunoconjugate of claim17, wherein said therapeutic agent is selected from the group consistingof a radionuclide, boron, gadolinium or uranium atoms, animmunomodulator, a cytokine, a hormone, a hormone antagonist, an enzyme,an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxic drug,a toxin, an angiogenesis inhibitor, a different antibody and acombination thereof.
 33. A therapeutic immunoconjugate of claim 32,wherein said cytotoxic agent is a drug or a toxin.
 34. A therapeuticimmunoconjugate of claim 33, wherein said drug is selected from thegroup consisting of antimitotic, alkylating, antimetabolite,angiogenesis- inhibiting, apoptotic, alkaloid, COX-2-inhibiting andantibiotic agents and combinations thereof.
 35. A therapeuticimmunoconjugate of claim 33, wherein said drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antibiotics, enzymes, epipodophyllotoxins, platinum coordinationcomplexes, vinca alkaloids, substituted ureas, methyl hydrazinederivatives, adrenocortical suppressants, hormone antagonists, enzymeinhibitors, endostatin, taxols and other taxanes, camptothecins,doxorubicins and their analogs, and a combination thereof.
 36. Atherapeutic immunoconjugate of claim 33, wherein said toxin is selectedfrom thegroup consisting of plant, microbial, and animal toxins, and asynthetic variation thereof.
 37. A therapeutic immunoconjugate of claim36, wherein said toxin is selected from the group consisting of ricin,abrin, alpha toxin, saporin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
 38. Atherapeutic immunoconjugate of claim 32, wherein said immunomodulator isselected from the group consisting of a cytokine, a stem cell growthfactor, a lymphotoxin, a hematopoietic factor, a colony stimulatingfactor (CSF), an interferon (IFN), a stem cell growth factor,erythropoietin, thrombopoietin, an antibody and a combination thereof.39. A therapeutic immunoconjugate of claim 38, wherein said lymphotoxinis tumor necrosis factor (TNF), said hematopoietic factor is aninterleukin (IL), said colony stimulating factor is granulocyte-colonystimulating factor (G-CSF) or granulocyte macrophage-colony stimulatingfactor (GM-CSF)), said interferon is interferons-a, -0 or -y, and saidstem cell growth factor is designated “S1 factor”. 40 A therapeuticimmunoconjugate of claim 38, wherein said cytokine is selected from thegroup consisting of IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-1 8,interferon-y, TNF-a and a combination thereof.
 41. The therapeuticimmunoconjugate of claim 32, wherein said radionuclide is selected fromthe group consisting of an Auger emitter, a beta-emitter and an alpha-emitter.
 42. A therapeutic immunoconjugate of claim 32, wherein saidradionuclide is selected from the group consisting of P-32, P-33, Sc-47,Fe-59, Cu-64, Cu-67, Se-75, As-77, Sr-89, Y-90, Mo-99, Rh-105, Pd-109,Ag-Il l , I-125, I-131, Pr-142, Pr-143, Pm-149, Sm-153, Th-161, Ho-166,Er-169, Lu-177, Re-186, Re-188, Re-189, Ir-194, Au-198, Au-199, Pb-211,Pb-212, and Bi-213, Co-58, Ga-67, Br-80m, Tc-99m, Rh- 103m, Pt-109,In-ill, Sb-l 19,1-125, Ho-161, Os-189m, Ir-192, Dy-152, At-211, Bi- 212,Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Fm-255and combinations thereof.
 43. The therapeutic immunoconjugate of claim32, wherein said Boron atom is B-10.
 44. The therapeutic immunoconjugateof claim 32, wherein said Gadolinium atom is Gd-157.
 45. The therapeuticimmunoconjugate of claim 32, wherein said Uranium atom is U-235.
 46. Atherapeutic immunoconjugate of claim 41,wherein said radionuclide has anenergy between 20 and 10,000 keV.
 47. A therapeutic immunoconjugate ofclaim 41,wherein said radionuclide is an Auger emitter and has an energyof less than 1000 keV.
 48. A therapeutic immunoconjugate of claim41,wherein said radionuclide is a P emitter and has an energy between 20and 5000 keV.
 49. A therapeutic immunoconjugate of claim 41, whereinsaid radionuclide is an a emitter and has an energy between 2000 and10,000 keV.
 50. A therapeutic immunoconjugate of claim 32, wherein saidphotoactive therapeutic agent is a chromogen or dye.
 51. Thediagnostic/detection or therapeutic immunoconjugate according to claim17, wherein said diagnostic/detection or therapeutic agent is bound tosaid MAb or fragment thereof by means of a carbohydrate moiety.
 52. Amultivalent, multispecific antibody or fragment thereof comprising oneor more antigen binding sites having affinity toward a AFP targetantigen and one or more hapten binding sites having affinity towardshapten molecules.
 53. The antibody or fragment thereof of claim 52,wherein said antibody or fragment thereof is humanized.
 54. The antibodyor fragment thereof of claim 52, wherein said antibody or fragmentthereof is a human antibody.
 55. The antibody or fragment thereof ofclaim 52, wherein said antibody or fragment thereof is chimerized. 56.The antibody or fragment thereof of claim 52, further comprising adiagnostic/detection or therapeutic agent.
 57. An antibody fusionprotein or fragment thereof comprising at least two AFP MAbs orfragments thereof, wherein said MAbs or fragments thereof are selectedfrom said MAb or fragment thereof of claim
 1. 58. An antibody fusionprotein or fragment thereof comprising at least one first AFP MAb orfragment thereof of claim 1 and at least one second MAb or fragmentthereof, other than the MAb or fragment thereof of any one of claim 1.59. The antibody fusion protein or fragment thereof of claim 57, furthercomprising a diagnostic/detection or therapeutic agent conjugated tosaid fusion protein or fragment thereof.
 60. The antibody fusion proteinor fragment thereof of claim 58, wherein said second MAb is acarcinoma-associated antibody.
 61. A method of treating a malignancy ina subject, comprising the step of administering to said subject atherapeutically effective amount of an antibody or fragment according toclaim 1, formulated in a pharmaceutically acceptable vehicle.
 62. Amethod of treating a malignancy in a subject, comprising the step ofadministering to said subject a therapeutically effective amount of aimmunoconjugate or fragment thereof of claim 17, formulated in apharmaceutically acceptable vehicle.
 63. A method ofdiagnosing/detecting a malignancy in a subject, comprising the step ofadministering to said subject a diagnostically effective amount of anantibody or fragment thereof of claim 1, formulated in apharmaceutically acceptable vehicle.
 64. A method ofdiagnosing/detecting a malignancy in a subject, comprising the step ofadministering to said subject a diagnostically effective amount of aimmunoconjugate or fragment thereof of claim 17, formulated in apharmaceutically acceptable vehicle.
 65. A method of treating ordiagnosing/detecting a malignancy in a subject, comprising the step ofadministering to said subject a therapeutically or diagnosticallyeffective amount of a fusion protein or fragment thereof of claim 57,formulated in a pharmaceutically acceptable vehicle.
 66. A method oftreating or diagnosing/detecting a malignancy in a subject, comprising(i) administering to a subject in need thereof the antibody or fragmentsthereof of claim 52; (ii) waiting a sufficient amount of time for anamount of the non- binding protein to clear the subject's bloodstream;and (iii) administering to said subject a carrier molecule comprising adiagnostic agent, a therapeutic agent, or a combination thereof, thatbinds to a binding site of said antibody.
 67. A DNA sequence comprisinga nucleic acid encoding an AFP MAb or fragment thereof selected from thegroup consisting (a) an AFP MAb or fragment thereof of claim 1; (b) anantibody fusion protein or fragment thereof comprising at least two ofsaid MAbs or fragments thereof; (c) an antibody fusion protein orfragment thereof comprising at least one first AFP MAb or fragmentthereof comprising said MAb or fragment thereof claim 1 and at least onesecond MAb or fragment thereof, other than the MAb or fragment thereofof claim 1; and (d) an antibody fusion protein or fragment thereofcomprising at least one first MAb or fragment thereof comprising saidMAb or fragment thereof of claim 1 and at least one second MAb orfragment thereof, other than the MAb or fragment thereof of claim Iwherein said second MAb is selected from the group consisting of CEA,EGP-1, EGP-2 (e.g., 17-1A), MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4,TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, Ep-CAM, Tn, andThomson-Friedenreich antigens, tumor necrosis antigens, tenascin, anoncogene, an oncogene product, IL-6, IGF-1, IGFR-1, tumor angiogenesisantigens, such as vascular endothelium growth factor (VEGF), placentalgrowth factor (PIGF), ED-B fibronectin, and other vascular growthfactors, ferritin, acidic isoferritin, Ga 733, or a combination thereof.68. An expression vector comprising the DNA sequence of claim
 67. 69. Ahost cell comprising the DNA sequence of claim
 67. 70. A method ofdelivering a diagnostic/detection or therapeutic agent, or a combinationthereof, to a target comprising (i) providing a composition comprisingan immunoconjugate that comprises the antibody or fragment thereof ofclaim 1 and (ii) administering to a subject in need thereof saidcomposition.
 71. The method of delivering of claim 70, wherein saiddiagnostic/detection agent comprises at least photoactive diagnosticagent.
 72. The method of delivering of claim 71, wherein saidphotoactive diagnostic agent comprises a chromagen or dye.
 73. Themethod of delivering of claim 70, wherein said diagnostic/detectionagent is a radionuclide with an energy between 20 and 2,000 keV.
 74. Themethod of delivering of claim 73, wherein said radionuclide is a gamma-,beta- or a positron-emitting isotope.
 75. The method of delivering ofclaim 73, wherein said radionuclide is selected from the groupconsisting of F-18, Mn-51, Mn-52m, Fe-52, Co-S5, Cu-62, Cu-64, Ga-68,As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-I 10, I-120, 1-124, Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97,Tc-99m, In- 111, In-114m, 1-123,1-125,1-131, Yb-169, Hg-197, and Tl-201.76. The method of delivering of claim 70, wherein said diagnostic agentis a contrast agent.
 77. The method of delivering of claim 76, whereinsaid contrast agent is a paramagnetic ion.
 78. The method of deliveringof claim 76, wherein said contrast agent is an ultrasound-enhancingagent.
 79. The method of delivering of claim 76, wherein said contrastagent is a radiopaque compound used in X-rays or computed tomography.80. The method of claim 79, wherein said radiopaque compound is selectedfrom the group consisting of iodine compounds, barium compounds, galliumcompounds and thallium compounds.
 81. The method of claim 79, whereinsaid radiopaque compound is selected from the group consisting ofbarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.
 82. The method of delivering of claim 78, wherein saidultrasound enhancing agent is a liposome that comprises a humanizedImmu31 or fragment thereof.
 83. The method of delivering of claim 82,wherein said liposome is gas-filled.
 84. The method of delivering ofclaim 77, wherein said paramagnetic ion is a metal comprising manganese,iron or gadolinium.
 85. The method of delivering of claim 70, whereinsaid therapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an enzyme inhibitor, a photoactive therapeutic agent, acytotoxic agent, and a combination thereof.
 86. The method of deliveringof claim 85, wherein said cytotoxic agent is a drug or a toxin.
 87. Themethod of delivering of claim 86, wherein said drug is selected from thegroup consisting of antimitotic, alkylating, antimetabolite,antiangiogenic, apoptotic, anthracyclines, alkaloid, COX-2-inhibitor andantibiotic agents, and combinations thereof.
 88. The method ofdelivering of claim 86, wherein said drug is selected from the groupconsisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antibiotics, enzymes, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormones, hormoneantagonists, endostatin, taxols, camptothecins, doxorubicins and theiranalogs, and a combination thereof.
 89. The method of claim 86, whereinsaid toxin is selected from the group consisting of plant, microbial andanimal toxin, and a synthetic variation thereof.
 90. The method ofdelivering of claim 86, wherein said toxin is selected from the groupconsisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase),DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,gelonin, diphtherin toxin, Pseudomonas exotoxin, and Pseudomonasendotoxin.
 91. The method of delivering of claim 85, wherein saidimmunomodulator is selected from the group consisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin, an antibody, and a combinationthereof.
 92. The method of delivering of claim 91, wherein saidlymphotoxin is tumor necrosis factor (TNF), said hematopoietic factor isan interleukin (IL), said colony stimulating factor isgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), said interferon isinterferons-a, - or -y, and said stem cell growth factor is designated“S1 factor”.
 93. The method of delivering of claim 91, wherein saidcytokine is selected from the group consisting of IL-1, IL-2, IL-3,IL-6, IL-1 0, IL-12, IL-1 8, interferon-y, TNF-(X and a combinationthereof.
 94. The method of delivering of claim 85, wherein saidradionuclide is selected from the group consisting of an Auger emitter,a 0 emitter and an a emitter.
 95. The method of delivering of claim 85,wherein said radionuclide is selected from the group consisting of P-32,P-33, Sc-47, Fe-59, Cu-64, Cu-67, Se-75, As-77, Sr-89, Y-90, Mo-99,Rh-105, Pd-109, Ag- II, I-125, 1-131, Pr-142, Pr-143, Pm-149, Sm-153,Th-161, Ho-166, Er-169, Lu-177, Re-186, Re-188, Re-189, Ir-194, Au-198,Au-199, Pb-211, Pb-212, and Bi-213, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m, Ir-192, Dy-152,At-211, Bi- 212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217,Bi-213, Fm-255 and combinations thereof.
 96. The method of delivering ofclaim 94,wherein said radionuclide has an energy between 20 and 10,000keV.
 97. The method of delivering of claim 94,wherein said radionuclideis an Auger emitter and has an energy of less than 1000 keV.
 98. Themethod of delivering of claim 94,wherein said radionuclide is a ,emitter and has an energy between 20 and 5000 keV.
 99. The method ofdelivering of claim 94, wherein said radionuclide is an a emitter andhas an energy between 2000 and 10,000 keV.
 100. The method of deliveringof claim 85, wherein said photoactive therapeutic agent is a chromogenor dye.
 101. A method of delivering a diagnostic/detection agent, atherapeutic agent, or a combination thereof to a target, comprising: (i)administering to a subject the antibody or fragments thereof of claim52; (ii) waiting a sufficient amount of time for an amount of thenon-binding protein to clear the subject's blood stream; and (iii)administering to said subject a carrier molecule comprising adiagnostic/detection agent, a therapeutic agent, or a combinationthereof, that binds to a binding site of said antibody.
 102. The methodof claim 101, wherein said carrier molecule binds to more than onebinding site of the antibody.
 103. The method of claim 101, wherein saiddiagnostic/detection agent or said therapeutic agent is selected fromthe group comprising isotopes, dyes, chromagens, contrast agents, drugs,toxins, cytokines, enzymes, enzyme inhibitors, hormones, hormoneantagonists, growth factors, radionuclides, and metals.
 104. A method oftreating a malignancy in a subject comprising administering to saidsubject a therapeutically effective amount of an antibody or fragmentthereof or an antibody fusion protein or fragment thereof comprising atleast two MAbs or fragments thereof, wherein at least one anti-AFP MAbor fragment thereof or fusion proteins or fragments thereof of claim 1formulated in a pharmaceutically suitable excipient.
 105. A method oftreating a malignancy in a subject comprising administering to saidsubject a therapeutically effective amount of an antibody or fragmentthereof comprising at least two MAbs or fragments thereof, comprisingthe MAb of claim 1, and formulated in a pharmaceutically suitableexcipient.
 106. The method of claim 104, further comprising a second Mabor fragment thereof not of claim
 1. 107. The method of claim 106,wherein said second Mab or fragment thereof is a naked Mab or fragmentthereof.
 108. The method of claim 106, wherein said second MAb orfragment thereof is selected from the group consisting of antibodiesagainst CEA, EGP-1, EGP-2 (e.g., 17-lA), MUC-1, MUC-2, MUC-3, MUC-4,PAM-4, KC4, TAG-72, EGFR, HER2/neu, BrE3, Le-Y, A3, Ep-CAM, Tn, andThomson-Friedenreich antigens, tumor necrosis antigens, ferritin, acidicisoferritin, tenascin, an oncogene, an oncogene product, IL-6, IGF-1,IGFR-1, tumor angiogenesis antigens, such as vascular endothelium growthfactor (VEGF), placental growth factor (PIGF), ED-B fibronectin, andother vascular growth factors, Ga 733, or a combination thereof. 109.The method of claim 108, wherein said second MAb is immunoconjugated toa therapeutic or diagnostic/detection agent.
 110. The method of claim104, wherein said anti-AFP antibody is administered parenterally. 111.The method of claim 110, wherein said anti-AFP antibody is administeredin a dosage of 20 to 2000 milligrams protein per dose.
 112. The methodof claim 110, wherein said dosage is repeatedly administered.
 113. Themethod of claim 104, wherein said anti-AFP antibody is selected from thegroup consisting of a subhuman primate anti-AFP antibody, murinemonoclonal anti-AFP antibody, chimeric anti-AFP antibody, human anti-AFPantibody, and humanized anti- AFP antibody.
 114. The method of claim113, wherein said chimeric, human and humanized anti-AFP antibodyconstant and hinge regions comprise constant and hinge regions of ahuman IgG1.
 115. The method of claim 104, wherein said anti-AFP antibodyor fragment thereof is administered before, in conjunction with, orafter a second conjugated antibody reactive with a second tumor markerexpressed by said malignancy is administered to said subject.
 116. Themethod of claim 104, wherein a first binding site of the anti-AFPantibody or fragment thereof is present in a multivalent, multispecificfusion protein or chemical conjugate and a second binding site isreactive with a tumor marker substance other than AFP.
 117. The methodof claim 104, wherein said anti-AFP antibody or fragment thereof isadministered before, concurrently, or after at least one therapeutic ordiagnostic/detection agent.
 118. The method of claim 117, wherein saidtherapeutic or diagnostic/detection agent is conjugated to an antibodythat targets a tumor marker that is expressed by said malignancy.
 119. Amethod of diagnosing or detecting a malignancy in a subject comprisingadministering to said subject a diagnostically effective amount of adiagnostic/detecting conjugate comprising a anti-AFP MAb or fragmentthereof or an fusion protein or fragment thereof of claim 1, whereinsaid anti-AFP MAb or fragment thereof or fusion protein or fragmentthereof is bound to at least one diagnostic/detection agent, formulatedin a pharmaceutically suitable excipient.
 120. A method of treating acancer cell in a subject comprising (i) administering to said subject atherapeutically effective amount of a composition comprising a naked orconjugated anti-AFP MAb or fragment thereof or antibody fusion proteinor fragment thereof, of claim 1, (ii) formulating said anti-AFP MAb orfragment thereof or antibody fusion protein or fragment thereof in apharmaceutically suitable excipient.
 121. The method of claim 120,wherein said composition further comprises a second antibody or fragmentthereof, or fusion protein or fragment thereof, not in claim
 1. 122. Themethod of claim 120, wherein said composition further comprises a secondantibody or fragment thereof or fusion protein or fragment thereof, ofclaim
 1. 123. The method of claim 121, wherein said antibody or fragmentthereof is a naked antibody or fragment thereof.
 124. The method ofclaim 121, wherein said second antibody or fragment thereof is selectedfrom the group consisting of antibodies to CEA, EGP-1, EGP-2 (e.g.,17-lA), MUC-1, MUC-2, MUC-3, MUC-4, PAM-4, KC4, TAG-72, EGFR, HER2/neu,BrE3, Le-Y, A3, Ep-CAM, Tn, and Thomson-Friedenreich antigens, tumornecrosis antigens, ferritin, acidic isoferritin, tenascin, an oncogene,an oncogene product, IL-6, IGF-1, IGFR-1, tumor angiogenesis antigens,such as vascular endothelium growth factor (VEGF), placental growthfactor (PIGF), ED-B fibronectin, and other vascular growth factors, Ga733, or a combination thereof.
 125. The method of claim 120, whereinsaid naked anti-AFP antibody is administered parenterally.
 126. Themethod of claim 125, wherein said naked anti-AFP antibody isadministered in a dosage of 20 to 2000 milligrams protein per dose. 127.The method of claim 126, wherein said dosage is repeatedly administered.128. The method of claim 120, wherein said naked anti-AFP antibody isselected from the group consisting of subhuman primate anti-AFPantibody, murine monoclonal anti-AFP antibody, chimeric anti-AFPantibody, human anti-AFP antibody, and humanized anti-AFP antibody. 129.The method of claim 128, wherein said chimeric, human and humanizednaked anti-AFP antibody constant and hinge regions comprise constant andhinge regions of a human IgGI.
 130. The method of claim 121, whereinsaid anti-AFP antibody is administered before, in conjunction with, orafter a second antibody reactive with a second tumor marker expressed bysaid malignancy is administered to said subject.
 131. The method ofclaim 120, wherein said anti-AFP antibody is administered before,concurrently or after a therapeutic or diagnostic/detection agent. 132.The method of claim 120, wherein said anti-AFP antibody is a nakedlmmu31 antibody.
 133. A method of diagnosing or detecting a malignancyin a subject comprising (i) performing an in vitro diagnosis assay on aspecimen from said subject with a composition comprising a anti-AFP MAbor fragment thereof or a antibody fusion protein or fragment thereof ofclaim
 1. 134. The method of claim 133, wherein said malignancy is acarcinoma expressing AFP.
 135. The method of claim 134, wherein saidcarcinoma is a hepatocellular carcinoma or a hepatoblastoma.
 136. Themethod of claim 135, wherein said carcinoma is a germ cell tumor. 137.The method of claim 133, wherein said in vitro diagnosis assay isselected from the group consisting of immunoassays, RT-PCR andimmunohistochemistry.
 138. The method of claim 137, wherein saiddiagnostic assay is RT-PCR or immunoassays.
 139. The method of claim138, wherein said specimen is body fluid or a tissue or cell population.140. The method of claim 137, wherein said diagnostic assay isimmunohistochemistry or immunocytochemistry.
 141. The method of claim140, wherein said specimen is a cell aliquot or a tissue.
 142. Themethod of claim 61 wherein said subject is a mammal.
 143. The method of142, wherein said subject is a human.
 144. The method of 142, whereinsaid subject is a domestic pet.
 145. The method of 142, wherein saidsubject is selected from the group consisting of a horse, dog, and cat.146. A method of treating or identifying diseased tissues in a subject,comprising: (A) administering to said subject a bi-specific antibody orantibody fragment having at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds atargetable conjugate, wherein said one arm that specificially binds atargeted tissue is an Immu31 antibody; (B) optionally, administering tosaid subject a clearing composition, and allowing said composition toclear non-localized antibodies or antibody fragments from circulation;(C) administering to said subject a first targetable conjugate whichcomprises a carrier portion which comprises or bears at least oneepitope recognizable by said at least one other arm of said bi-specificantibody or antibody fragment, and one or more conjugated therapeutic ordiagnostic agents; and (D) when said therapeutic agent is an enzyme,further administering to said subject 1) a prodrug, when said enzyme iscapable of converting said prodrug to a drug at the target site; or 2) adrug which is capable of being detoxified in said subject to form anintermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 3) a prodrug which is activated in said subject through naturalprocesses and is subject to detoxification by conversion to anintermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 4) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by said atleast one other arm of said bi-specific antibody or antibody fragment,and a prodrug, when said enzyme is capable of converting said prodrug toa drug at the target site.
 147. The method of claim 146, wherein saidtargetable conjugate comprises at least two HSG haptens.
 148. The methodof claim 146, further comprising, when said first targetable conjugatecomprises a prodrug, administering a second targetable conjugate whichcomprises a carrier portion which comprises or bears at least oneepitope recognizable by said at least one other arm of said bi-specificantibody or antibody fragment, and an enzyme capable of converting saidprodrug to a drug or of reconverting a detoxified intermediate of saiddrug to a toxic form.
 149. The method of claim 146, wherein saiddiagnostic/detection agent is a radionuclide.
 150. The method of claim146, wherein said diagnostic/detection agent is a radionuclide with anenergy between 20 and 2,000 keV.
 151. The method of claim 149, whereinsaid diagnostic/detection agent emits 25-600 keV gamma particles and/orpositrons.
 152. The method of claim 146, wherein saiddiagnostic/detection agent comprises at least one photoactive diagnosticagent.
 153. The method of claim 152, wherein said photoactivediagnostic/detection agent comprises a chromagen or dye.
 154. The methodof claim 146, wherein said diagnostic/detection agent is a radiopaquecompound.
 155. The method of claim 149, wherein said radionuclide is agamma-, beta- or a positron-emitting isotope.
 156. The method of claim155, wherein said radionuclide is selected from the group consisting ofF-1 8, Mn-51, Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68, As- 72, Br-75,Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-110, I-120, 1-124, Cr-51,Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, Ru-97, Tc-99m, In-I1, In-114m,I-1 23, 1-125, 1-131, Yb-169, Hg-197, and Tl-201.
 157. The method ofclaim 146, wherein said diagnostic/detection agent is a contrast agent.158. The method of claim 157, wherein said contrast agent is aparamagnetic ion.
 159. The method of claim 154, wherein said radiopaquecompound is selected from the group consisting of iodine compounds,barium compounds, gallium compounds and thallium compounds.
 160. Themethod of claim 159, wherein said radiopaque compound is selected fromthe group consisting of barium, diatrizoate, ethiodized oil, galliumcitrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamicacid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid,iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid,iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid,ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate,propyliodone, and thallous chloride.
 161. The method of claim 157,wherein said contrast agent is an ultrasound enhancing agent.
 162. Themethod of claim 161, wherein said ultrasound enhancing agent is aliposome that is conjugated humanized, chimerized, or fully human lrmu31antibody or fragment thereof.
 163. The method of claim 162, wherein saidliposome is gas filled.
 164. A method of claim 158, wherein saidparamagnetic ion comprises a metal selected from the group consisting ofmanganese, iron and gadolinium.
 165. The method of claim 146, whereinsaid therapeutic agent is selected from the group consisting of aradionuclide, boron, gadolinium or uranium atoms, an immunomodulator, ahormone, a hormone antagonist, an enzyme, and enzyme inhibitor, aphotoactive therapeutic agent, a cytotoxic agent, an angiogenesisinhibitor, and a combination thereof.
 166. The method of claim 165,wherein said cytotoxic agent is a drug, prodrug or a toxin.
 167. Themethod of claim 166, wherein said prodrug is selected from the groupconsisting of epirubicin glucuronide, CPT- 11, etoposide glucuronide,daunomicin glucuronide and doxorubicin glucuronide.
 168. The method ofclaim 166, wherein said toxin is selected from the group consisting of aplant toxin, an animal toxin and a microbial toxin and a syntheticvariation thereof.
 169. The method of claim 166, wherein said toxin isselected from the group consisting of ricin, abrin, ribonuclease(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtherin toxin, Pseudomonas exotoxin, andPseudomonas endotoxin.
 170. The method of claim 166, wherein said drugis selected from the group consisting of antimitotic, alkylating,antimetabolite, angiogenesis-inhibiting, apoptotic, alkaloid,COX-2-inhibiting and antibiotic agents and combinations thereof. 171.The method of claim 166, wherein said drug is selected from the groupconsisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines,taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs,antibiotics, enzymes, epipodophyllotoxins, platinum coordinationcomplexes, vinca alkaloids, substituted ureas, methyl hydrazinederivatives, adrenocortical suppressants, hormones, hormone antagonistsendostatin, taxols, camptothecins, doxorubicins and their analogs, and acombination thereof.
 172. The method of claim 165, wherein saidimmunomodulator is selected from the groupconsisting of a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin, an antibody agonist orantagonist to an immunomodulator, and a combination thereof.
 173. Themethod of claim 172, wherein said lymphotoxin is tumor necrosis factor(TNF), said hematopoietic factor is an interleukin (IL), said colonystimulating factor is granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage- colony stimulating factor (GM-CSF)), saidinterferon is interferons-oa, -, or -y, and said stem cell growth factoris designated “S1 factor”.
 174. Themethod of claim 172, wherein saidcytokine is selected from the group consisting of IL-1, IL-2, IL-3,IL-6, IL-10, IL-12, IL-1 8, interferon-y, TNF-A and a combinationthereof.
 175. The method of claim 165, wherein said radionuclide isselected from the group consisting of an Auger emitter, a P emitter andan a emitter.
 176. The method of claim 165, wherein said radionuclide isselected from the group consisting of P-32, P-33, Sc-47, Fe-59, Cu-64,Cu-67, Se-75, As-77, Sr-89, Y- 9 0, Mo-99, Rh-105, Pd-109, Ag-i Il,I-125, 1-131, Pr-142, Pr-143, Pm-149, Sm-153, Tb- 161, Ho-166, Er-169,Lu-1 77, Re-186, Re-188, Re-189, Ir-194, Au-198, Au-l99, Pb-211, Pb-212,and Bi-213, Co- 58, Ga-67, Br-80m, Tc-99m, Rh-i 03m, Pt-1 09, In- 111,Sb-i 19,1-125, Ho-161, Os-189m, Ir-192, Dy-152, At-211, Bi-212, Ra-223,Rn- 219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Fm-255 andcombinations thereof.
 177. The method of claim 165, wherein said Boronatom is B-i
 0. 178. The method of claim 165, wherein said Gadoliniumatom is Gd-157.
 179. The method of claim 165, wherein said Uranium atomis U-235.
 180. The method of claim 175,wherein said radionuclide has anenergy between 20 and 10,000 keV.
 181. The method of claim 175,whereinsaid radionuclide is an Auger emitter and has an energy of less than1000 keV.
 182. The method of claim 175,wherein said radionuclide is a Pemitter and has an energy between 20 and 5000 keV.
 183. The method ofclaim 175, wherein said radionuclide is an a emitter and has an energybetween 2000 and 10,000 keV.
 184. The method of claim 146, wherein saidtargetable conjugate comprises one or more radioactive isotopes usefulfor killing diseased tissue.
 185. The method of claim 177, wherein saidtargetable conjugate comprises 'OB atoms, and said method furthercomprises the step of irradiating said boron atoms localized at saiddiseased tissue, thereby effecting BNCT of said diseased tissue. 186.The method of claim 146, wherein the targetable conjugate comprises oneor more agents for photodynamic therapy.
 187. The method of claim 186,wherein said agent for photodynamic therapy is a photosensitizer. 188.The method of claim 187, wherein said photosensitizer is selected fromthe group consisting of benzoporphyrin monoacid ring A (BPD-MA), tinetiopurpurin (SnET2), sulfonated aluminum phthalocyanine (AlSPc) andlutetium texaphyrin (Lutex).
 189. The method of claim 146, wherein saidat least one arm that specifically binds a targeted tissue is a human,chimeric or humanized lmmu31 antibody or a fragment of a human, chimericor humanized Immu31 antibody.
 190. The method of claim 146, wherein saidat least one other arm that specifically binds a targetable conjugate isa human, chimeric or humanized lrmu31 antibody or a fragment of a human,chimeric or humanized lrmu31 antibody.
 191. The method of claim 146,wherein said targeted tissue is a tumor.
 192. The method of claim 191,wherein said tumor produces or is associated with alpha-fetoprotein(AFP).
 193. The method of claim 146, wherein said lmmu31 antibody orfragment thereof comprises the Fv of MAb lmmu3
 1. 194. The method ofclaim 146, wherein said bispecific antibody is a fusion protein. 195.The method of claim 194, wherein the fusion protein is trivalent, andincorporates the Fv of an antibody reactive with AFP.
 196. A method fordetecting or treating tumors expressing AFP in a mammal, comprising: (A)administering an effective amount of a bispecific antibody or antibodyfragment comprising at least one arm that specifically binds a targetedtissue and at least one other arm that specifically binds a targetableconjugate, wherein said one arm that specifically binds a targetedtissue is an lmmu31 antibody or fragment thereof, and (B) administeringa targetable conjugate selected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


197. A method of claim 196, further comprising administering to saidsubject a clearing composition, and allowing said composition to clearnon-localized antibodies or antibody fragments from circulation.
 198. Akit useful for treating or identifying diseased tissues in a subjectcomprising: (A) a bi-specific antibody or antibody fragment having atleast one arm that specifically binds a targeted tissue and at least oneother arm that specifically binds a targetable conjugate, wherein saidone arm that specifically binds a targeted tissue is an lmmu3 l antibodyor fragment thereof; (B) a first targetable conjugate which comprises acarrier portion which comprises or bears at least one epitoperecognizable by said at least one other arm of said bi-specific antibodyor antibody fragment, and one or more conjugated therapeutic ordiagnostic agents; and (C) optionally, a clearing composition useful forclearing non-localized antibodies and antibody fragments; and (D)optionally, when said therapeutic agent conjugated to said firsttargetable conjugate is an enzyme, 1) a prodrug, when said enzyme iscapable of converting said prodrug to a drug at the target site; or 2) adrug which is capable of being detoxified in said subject to form anintermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or 3) a prodrug which is activated in said subject through naturalprocesses and is subject to detoxification by conversion to anintermediate of lower toxicity, when said enzyme is capable ofreconverting said detoxified intermediate to a toxic form, and,therefore, of increasing the toxicity of said drug at the target site,or. 4) a second targetable conjugate which comprises a carrier portionwhich comprises or bears at least one epitope recognizable by said atleast one other arm of said bi-specific antibody or antibody fragment,and a prodrug, when said enzyme is capable of converting said prodrug toa drug at the target site.
 199. The kit of claim 198, wherein saidtargetable conjugate is selected from the group consisting of: (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


200. A method of screening for a targetable conjugate comprising: (A)contacting said targetable construct with a bi-specific antibody orantibody fragment having at least one arm that specifically binds atargeted tissue and at least one other arm that specifically binds saidtargetable conjugate to give a mixture, wherein said one arm thatspecifically binds a targeted tissue is a Inmu3 1 antibody or fragmentthereof; and (B) optionally incubating said mixture; and (C) analyzingsaid mixture.
 201. A method for imaging malignant tissue or cells in amammal expressing AFP, comprising: (A) administering an effective amountof a bispecific antibody or antibody fragment comprising at least onearm that specifically binds a targeted tissue and at least one other armthat specifically binds a targetable conjugate, wherein said one armthat specifically binds a targeted tissue is an Immu3 1 antibody orfragment thereof; and (B) administering a targetable conjugate selectedfrom the group consisting of (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂;(ii) DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


202. A method of intraoperatively identifying/disclosing diseasedtissues expressing AFP, in a subject, comprising: (A) administering aneffective amount of a bispecific antibody or antibody fragmentcomprising at least one arm that specifically binds a targeted tissueexpressing AFP and at least one other arm that specifically binds atargetable conjugate, wherein said one arm that specifically binds atargeted tissue is an fIrnu3 1 antibody or fragment thereof; and (B)administering a targetable conjugate selected from the group consistingof (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


203. A method for the endoscopic identification of diseased tissuesexpressing AFP, in-a subject, comprising: (A) administering an effectiveamount of a bispecific antibody or antibody fragment comprising at leastone arm that specifically binds a targeted tissue expressing AFP and atleast one other arm that specifically binds a targetable conjugatewherein said one arm that specifically binds a targeted tissue is aImmu3 1 antibody or fragment thereof; and (B) administering a targetableconjugate selected from the group consisting of (i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


204. A method for the intravascular identification of diseased tissuesexpressing AFP, in a subject, comprising: (A) administering aneffective. amount of a bispecific antibody or antibody fragmentcomprising at least one arm that specifically binds a targeted tissueexpressing AFP and at least one other arm that specifically binds atargetable conjugate wherein said one arm that specifically binds atargeted tissue is a hmmu3 1 antibody or fragment thereof; and (B)administering a targetable conjugate selected from the group consistingof (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂; (ii)DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂; (iii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH₂;


205. A method of detection of lesions during an endoscopic,laparoscopic, intravascular catheter, or surgical procedure, wherein themethod comprises: (A) injecting a subject who is to undergo such aprocedure with a bispecific antibody F(ab)₂ or F(ab')₂ fragment, whereinthe bispecific antibody or fragment has a first antibody binding sitewhich specifically binds to a AFP antigen, and has a second antibodybinding site which specifically binds to a hapten, and permitting theantibody fragment to accrete at target sites; (B) optionally clearingnon-targeted antibody fragments using a galactosylated anti-idiotypeclearing agent if the bispecific fragment is not largely cleared fromcirculation within about 24 hours of injection, and injecting a bivalentlabeled hapten, which quickly localizes at the target site and clearsthrough the kidneys; (C) detecting the presence of the hapten byclose-range detection of elevated levels of accreted label at the targetsites with detection means, within 48 hours of the first injection, andconducting said procedure, wherein said detection is performed withoutthe use of a contrast agent or subtraction agent.
 206. The method ofclaim 205, wherein said hapten is labeled with a diagnostic/detectionradioisotope, a MRI image-enhancing agent or a fluorescent label.
 207. Amethod for close-range lesion detection, during an operative,intravascular, laparoscopic, or endoscopic procedure, wherein the methodcomprises: (A) injecting a subject to such a procedure parenterally withan effective amount of an lmmu31 immunoconjugate or fragment thereof,(B) conducting the procedure within 48 hours of the injection; (C)scanning the accessed interior of the subject at close range with adetection means for detecting the presence of said labeled antibody orfragment thereof; and (D) locating the sites of accretion of saidlabeled antibody or fragment thereof by detecting elevated levels ofsaid labeled antibody or fragment thereof at such sites with thedetection means.
 208. The method of claim 207, wherein said immu31immunoconjugate or fragment thereof comprises a radioisotope that emitsat an energy of 20-1,000 keV.
 209. The method of claim 208, wherein theradioisotope is selected from the group consisting of technetium-99m,iodine-125, iodine-131, iodine-123, indium-11, fluorine-18, gallium 68and gallium-67.
 210. The method of claim 209, wherein irnmu31immunoconjugate or fragment thereof comprises a non-isotopic agent. 211.The method of claim 210, wherein said non-isotopic agent is aphotoactive agent.
 212. The immunoconjugate of claim 17, wherein saidtherapeutic agent is a ribonuclease.
 213. The immunoconjugate of claim212, wherein said therapeutic agent is onconase.